Dendrimer based nanodevices for therapeutic and imaging purposes

ABSTRACT

A nanodevice composition including N-acetyl cysteine linked to a dendrimer, such as a PAMAM dendrimer or a multiarm PEG polymer, is provided. Also provided is a nanodevice for targeted delivery of a compound to a location in need of treatment. The nanodevice includes a PAMAM dendrimer or multiarm PEG polymer, linked to the compound via a disulfide bond. There is provided a nanodevice composition for localizing and delivering therapeutically active agents, the nanodevice includes a PAMAM dendrimer or multiarm PEG polymer and at least one therapeutically active agent attached to the PAMAM dendrimer or multiarm PEG polymer. A method of site-specific delivery of a therapeutically active agent, by attaching a therapeutically active agent to a PAMAM dendrimer or multiarm PEG polymer using a disulfide bond, administering the PAMAM dendrimer or multiarm PEG polymer to a patient in need of treatment, localizing the dendrimer or multiarm PEG polymer to a site in need of treatment, and releasing the therapeutically active agent at the site in need of treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/477,725, filed Sep. 4, 2014, which is a continuation of U.S. patentapplication Ser. No. 12/797,657, filed Jun. 10, 2010, which claimspriority to U.S. Provisional Patent Application No. 61/319,285, filedMar. 31, 2010 and U.S. Provisional Patent Application No. 61/187,263,filed Jun. 15, 2009 all of which are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Apr. 28, 2010 as a text file named“ATL.WSU_09-945_CON(2)_ST25.txt,” created on Apr. 16, 2019, and having asize of 2,154 bytes is hereby incorporated by reference pursuant to 37C.F.R. § 1.52(e)(5).

BACKGROUND ART 1. Field of the Invention

Generally, the present invention relates to therapeutic nanodevicesbased on dendritic polymers. More specifically, the present inventionrelates to nanodevices for use in treating neuroinflammation andinfections in maternal-fetal medicine.

2. Description of Related Art

Currently, there is a need to develop techniques and compounds that areable to effectively deliver bioactive agents to cells. While there arenumerous systems under review for effectuating the delivery, theproblems surrounding the delivery mechanisms have outweighed theusefulness of the systems. Examples of such systems include viraltransfection systems and non-viral transfection systems. Viral systemstypically have higher transfection efficiency than non-viral systems,but there have been questions regarding the safety of viral systems. Inaddition, viral vector preparation tends to be a complicated andexpensive process. Although non-viral transfection systems generally areless efficient than viral systems, they have received significantattention because they are generally believed to be safer and easier toprepare than viral systems.

A number of non-viral transfection systems involve the use of cationicpolymers that are complexed to bioactive agents. Examples of cationicpolymers that have been used as gene carriers include poly(L-lysine)(PLL), polyethyleneimine (PEI), chitosan, PAMAM dendrimers, andpoly(2-dimethylamino)ethyl methacrylate (pDMAEMA). Unfortunately,transfection efficiency is typically poor with PLL, and high molecularweight PLL has shown significant toxicity to cells. Unfortunately,PEI-dendrimers have been reported to be toxic to cells, thus limitingthe potential for using PEI as a gene delivery tool in applications tohuman patients.

Dendrimers, as the term is used herein, are a class of polymers oftencalled starburst polymers because of their shape. These dendrimers havea molecular architecture with an interior core, interior layers (or“generations”) of repeating units regularly attached to this interiorcore, and an exterior surface of terminal groups attached to theoutermost generation. These starburst polymers are radially symmetricaland have a branched or tree-like structure. The number of generationscan be controlled by the conditions of manufacture, leading to differentsize molecules having different numbers of terminal groups. U.S. Pat.No. 4,587,329 entitled Dense Star Polymers Having Two DimensionalMolecular Diameter, issued May 6, 1986 to the Dow Chemical Company, thedisclosure of which is incorporated by reference, describes thesestarburst dendrimers and methods of their manufacture. These starburstdendrimers can be made to exact, repeatable molecular weights with thesame number of functional groups on each dendrimer. These functionalgroups can react with a material to be carried, such as a pharmaceuticalor agricultural product, or the material to be carried can be associatedwith this dendrimer in a non-reactive manner.

One family of dendrimers is based on an amidoamine repeat structure,forming what are known as poly(amidoamine) dendrimers (“PAMAM”). PAMAMdendrimers are grown from an amine containing core structure such asethylene diamine, or the like. Normally ethylene diamine is used as thecore or initiator of the reaction. The basic synthesis for PAMAMstarburst dendrimers begins with ethylene diamine (EDA) being reactedwith methyl acrylate under control conditions such that a Michaeladdition of one molecule of EDA to four molecules of methyl acrylateoccurs. This forms the initiator or core adduct. Following the removalof excess methyl acrylate, the core adduct is reacted with an excess ofEDA to form a 0 generation molecule having four amidoamine groups. Theexcess EDA is removed and the 0 generation molecule can be reacted withmethyl acrylate in another Michael addition reaction to form a firstgeneration molecule containing eight primary amine groups. Acontinuation of this stepwise procedure forms the other generations insequence.

These delivery systems are being developed to increase thebioavailability of the bioactive agents that are administered. Thebioavailability of many compositions is limited when the compound isadministered orally. This low bioavailability is often due to incompleteabsorption and first-pass metabolism of the compounds. Additionally,rapid degradation of antioxidants in the body fluid and elimination ofantioxidants from the body further decreases the beneficial effects ofantioxidants. Further, some compounds may be limited by theirstoichiometric quantities. By combining the compounds with a dendrimerthe goal is to overcome these problems. However, as stated above,currently available systems have met with little to no success. It wouldtherefore be useful to develop a delivery system that both overcomes theproblems outlined above as well as increasing the bioavailability of theadministered compounds.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a nanodevicecomposition including N-acetyl cysteine linked to a PAMAM dendrimer or amultiarm PEG (polyethylene glycol) polymer.

Also provided is a nanodevice for targeted delivery of a compound to alocation in need of treatment. The nanodevice includes a PAMAM dendrimeror or a multiarm PEG (polyethylene glycol) polymer linked to thecompound via a disulfide, amide, or ester bond. There is provided ananodevice composition for localizing and delivering therapeuticallyactive agents, the nanodevice includes a PAMAM dendrimer or or amultiarm PEG and at least one therapeutically active agent attached tothe PAMAM dendrimer or said multiarm PEG.

A method of site-specific delivery of a therapeutically active agent, byattaching a therapeutically active agent to a PAMAM dendrimer or or amultiarm PEG using a disulfide bond, administering the PAMAM dendrimeror or a multiarm PEG to a patient in need of treatment, localizing thedendrimer or or a multiarm PEG to a site in need of treatment, andreleasing the therapeutically active agent at the site in need oftreatment is further provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts N-Acetyl Cysteine (NAC) linked to G4-PAMAM-NH₂ dendrimerby disulfide bond using the spacer SPDP such that the bond between NACand SPDP appended on dendrimer surface is disulfide.

FIG. 2 depicts N-Acetyl Cysteine (NAC) linked to G4-PAMAM-COOH dendrimerby disulfide bond using the spacer Glutathione (GSH) such that the bondbetween NAC and GSH on appended on dendrimer surface is disulfide.

FIG. 3 depicts N-Acetyl Cysteine (NAC) linked to G4-PAMAM-OH dendrimersby disulfide bond using the two spacer molecules, Gamma-aminobutyricacid (GABA) and SPDP such that the bond between NAC and SPDP appended ondendrimer surface is disulfide

FIG. 4 depicts Ampicillin linked to G4-PAMAM-OH dendrimers by disulfidebond using the two spacer molecules, Gamma-aminobutyric acid (GABA) andSPDP such that the bond between Ampicillin and SPDP appended ondendrimer surface is disulfide.

FIG. 5 depicts Doxycycline linked to G4-PAMAM-OH dendrimer by disulfidebond using the spacer SPDP such that the bond between doxycycline andSPDP appended on dendrimer surface is disulfide.

FIG. 6 depicts Dexamethasone linked to G4-PAMAM-OH dendrimers bydisulfide bond using the three spacer molecules, Gamma-aminobutyric acid(GABA), SPDP and SPDP such that the bond between Dexamethasone and SPDPappended on dendrimer surface is disulfide.

FIG. 7 depicts Indomethacin linked to G4-PAMAM-OH dendrimers bydisulfide bond using the three spacer molecules, Gamma-aminobutyric acid(GABA), SPDP and SPDP such that the bond between Indomethacin and SPDPappended on dendrimer surface is disulfide.

FIG. 8 depicts Progesterone linked to G4-PAMAM-OH dendrimers bydisulfide bond using the two spacer molecules of SPDP linked to eachother such that the bond between Progesterone and SPDP appended ondendrimer surface is disulfide.

FIG. 9 depicts 5¹Adenine-GUCGGAGGCUUAAUUACA-3¹ (SEQ ID NO: 1) nucleotidelinked to G4-PAMAM-OH dendrimers by disulfide bond using four spacermolecules of linked in order GABA-SPDP-SPDP-GABA linked to each othersuch that the bond between Adenine nucleotide and SPDP-GABA appended ondendrimer surface is disulfide.

FIG. 10 depicts 5¹Cytosine-AGGAAAUUUGCCUAUUGA-3¹ (SEQ ID NO: 2)nucleotide linked to G4-PAMAM-OH dendrimers by disulfide bond using fourspacer molecules of linked in order GABA-SPDP-SPDP-GABA linked to eachother such that the bond between Cytosine nucleotide and SPDP-GABAappended on dendrimer surface is disulfide.

FIG. 11 depicts 5¹Uracil-AAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3) nucleotidelinked to G4-PAMAM-OH dendrimers by disulfide bond using four spacermolecules of linked in order GABA-SPDP-SPDP-GABA linked to each othersuch that the bond between Uracil nucleotide and SPDP-GABA appended ondendrimer surface is disulfide.

FIG. 12 shows the rapid release of NAC from 6 arm-PEG-S—S-NAC

FIG. 13 shows the rapid release of NAC from 8 arm-PEG-S—S-NAC

FIG. 14 shows an ROS assay. (A) BV-2 cells (passage 22) were co-treatedwith 100 ng/mL of LPS and the indicated concentration of NAC, PEG-NACconjugate 1 and its corresponding PEG for 72 hours. (B) BV-2 cells wereco-treated with 100 ng/mL of LPS and the indicated concentration of NAC,PEG-NAC conjugate 2 and its corresponding PEG for 72 hours. The amountof ROS released into the media was measured using Amplex Red.

FIG. 15 shows a NO release assay. (A) BV-2 cells (passage 22) wereco-treated with 100 ng/mL of LPS and the indicated concentration of NAC,PEG-NAC conjugate land its corresponding PEG for 72 hours. (B) BV-2cells were co-treated with 100 ng/mL of LPS and the indicatedconcentration of NAC, PEG-NAC conjugate 2 its corresponding PEG for 72hours. Nitrite in culture medium was measured using Griess reagentsystem.

FIG. 16 shows an TNF-α release assay. (A) BV-2 cells (passage 22) wereco-treated with 100 ng/mL of LPS and the indicated concentration of NAC,6-Arm-PEG-S—S-NAC conjugate (1) and its correspond 6-Arm-PEG-SH for 72hours (B) BV-2 cells were co-treated with 100 ng/mL of LPS and theindicated concentration of NAC, 8-Arm-PEG-S—S-NAC conjugate (3) and itscorrespond PEG for 72 hours. Three samples were in each group. TNF-α inculture medium was measured using mouse TNF-α ELISA Kit.

FIG. 17 shows the neurobehavioral scoring of rabbits treated with NAC,G4-PAMAM-NAC conjugate and endotoxin treated rabbits.

FIG. 18 shows the transport of G4-PAMAM-FITC across the rabbit amnioticmembrane (in-vitro evaluation).

FIG. 19 depicts the permeation coefficient determination forG4-PAMAM-FITC across the rabbit amniotic membrane (in vitro evaluation).

FIG. 20 shows the NF-κB protein expression.

FIG. 21 shows the NT-3 expression indicated oxidative injury.

FIG. 22 shows the mRNA expression of TNF-α and IL-6 in the brain.

FIG. 23 shows the GSH qualification in the hippocampus.

FIG. 24 shows the Uptake of FITC-G4OH in activated microglial cellsthrough subdural injection.

FIG. 25 depicts the biodistribution of FITC-G4OH in endotoxin exposedkits (SD Injection). Rabbit kits exposed to maternal inflammationG4OH-FITC localizes to activated microglial cells and astrocytes in thebrain far removed from the site of injection.

FIG. 26 depicts the biodistribution of FITC-G4OH after subduralinjection in controls. G4OH-FITC is taken up by some microglial cellsalong the lateral ventricle in control kits while no uptake is seen inastrocytes.

FIG. 27 depicts the biodistribution of FITC-G4OH following intravenousinjection. Dendrimer-FITC localizes in activated microglia andastrocytes on intravenous injection in endotoxin kits but not incontrols.

FIG. 28 depicts the brain uptake of G4OH—Cu[64] in fetalneuroinflammation using PET imaging. Increased uptake of G4OH—Cu[64] wasnoted in the newborn rabbit kits exposed to maternal inflammation.

FIG. 29 shows a library of D-NAC nanodevices.

FIG. 30 show polymers, linkers, and drug release times.

FIG. 31 shows the efficacy of Dendrimer-NAC Conjugates for suppressionof neuroinflammation (protein level). Dendrimer-NAC Conjugates result in10-100 greater suppression of NF-kB protein expression in thehippocampus of 5 day old rabbits exposed to maternal inflammation inutero (n=3-4 pups/group).

FIG. 32 shows the dendrimer-NAC Conjugates for suppression ofneuroinflammation (RNA level). The dendrimer nanodevice is 10-100 timesmore effective in suppressing mRNA expression of TNF-alpha in thehippocampus of endotoxin kits at 5 days of age (n=3 pups/group).

FIG. 33 show the dendrimer-NAC Conjugates for suppression ofneuroinflammation (RNA level). The dendrimer nanodevice is 10-100 timesmore effective than the free drug for suppression of IL-6 in thehippocampus (n=3 pups/group).

FIG. 34 shows the therapeutic Efficacy of Dendrimer-NAC Conjugates(effects on myelination). Treatment with dendrimer-NAC results inincreased myelination and better organization of myelin tracts whencompared to the drug alone.

FIG. 35 depicts the Maternal Infection and FIRS.

FIG. 36 depicts the mechanism of Brain Injury.

FIG. 37 depicts neonatal white matter damage and cerebral palsy.

FIG. 38 shows the clinical translation-PET imaging babies. PK11195imaging of neonate born to mother with severe chorioamnionitis withfunisitis at GA 28 5/7. Patient was asymptomatic at birth. Arrow pointsto increased tracer uptake in the periventricular regions.

FIG. 39 shows the biodistribution of dendrimers administered viasubdural injection. Dendrimers preferentially localize in activatedmicroglia and astrocytes in the endotoxin kits but not in the controls.No localization seen in neuronal cells.

FIG. 40 depicts the control administered via subdural injectionmicroglial (lectin) staining.

FIG. 41 depicts subdural injection astrocytes (GFAP staining).

FIG. 42 shows the biodistribution of dendrimers administered viaintravenous (IV). Dendrimers were seen in activated microglia andastrocytes in endotoxin kits and not in controls following IVadministration.

FIG. 43 shows the brain uptake of Dendrimer-⁶⁴Cu[64] in fetalneuroinflammation via PET imaging. Increased uptake of G4OH-⁶⁴Cu wasnoted in the newborn rabbits kits exposed to maternal inflammation.

FIG. 44 shows a synthesis scheme for PAMAM-OH-NAC.

FIG. 45 shows the in vitro release in the presence of GSH (pH=7.4).G4-PAMAM-NH₂—CO-Ethyl-S—S-NAC conjugate was dissolved in PBS at 1 mg/mlconcentration. G4-PAMAM-NH₂—CO-Ethyl-S—S-NAC conjugate release mediacontained 730 μM NAC in conjugated form. More than 60% of the drug wasreleased in less than two hours at intracellular GSH levels.

FIG. 46 shows in vitro efficacy: NO release assay. Even at the lowestdoses, dendrimer nanodevices showed better efficacy than free NAC at thehighest doses. Similar results for ROS, TNF-alpha, GSH depletion assays.

FIG. 47 depicts a rabbit model (neurobehavioral assessment) showingphenotype change upon dendrimer treatment. All endotoxin treated animalslooked the same on day 1. Control had no disease. Endo had PBStreatment. Free drug administered at 100 mg/kg NAC. Dendrimeradministered at 1 mg/kg NAC and 10 mg/kg NAC.

FIG. 48 depicts a rabbit model (neurobehavioral assessment). On day one,one injection was administered. Control had no disease. Endo had PBStreatment. Free drug administered at 100 mg/kg NAC. Dendrimeradministered at 1 mg/kg NAC and 10 mg/kg NAC.

FIG. 49 shows real-time RT-PCR Assays: Fetal brain.

FIG. 50 shows a dendrimer-NAC conjugates in vivo. 1 mg/kg of D-NAC wasas effective as 100 mg/kg of NAC alone in suppressing NF-κB. Endotoxinkits were treated with a single dose of NAC or 1/10^(th) or 1/100^(th)the dose of D-NAC on PND-1. Kits euthanized on PND 5 and NF-κBexpression in hippocampus determined.

FIG. 51 shows an ¹H NMR spectrum of the PAMAM-(COOH)₄₆-(NAC)₁₈conjugate. The appearance of methyl protons at 1.70, 1.92 ppm indicatingthe formation of GS-NAC conjugates with dendrimer.

FIG. 52 shows an ROS assay. BV-2 cells (passage 16) were stimulated with100 ng/mL of LPS for 24 hours and 72 hours after 3 hours pre-treatmentwith the indicated concentration of NAC, PAMAM-(COOH)₄₆-(NAC)₁₈conjugate and the corresponding amount of free dendrimer. Three sampleswere used for each group. The amount of ROS released into the media wasmeasured using Amplex Red. *P<0.05, **P<0.01 vs. group of LPS; ♦P<0.05,♦♦P<0.01 vs. group of NAC in same concentration.

FIG. 53 shows an NO release assay. BV-2 cells (passage 16) werestimulated with 100 ng/mL of LPS for 24 and 72 hours after 3 hourspre-treatment with the indicated concentration of NAC,PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate and the corresponding amount of freedendrimer. Three samples were used for each group. Nitrite in culturemedium was measured using Griess reagent system. *P<0.05, **P<0.01 vs.group of LPS; ♦P<0.05, ♦♦P<0.01 vs. group of NAC in same concentration.

FIG. 54 shows the effect of dendrimer on NO release. BV-2 cells (passage16) were stimulated with 100 ng/mL of LPS for 24 hours and 72 hoursafter 3 hours pre-treatment with the indicated concentration ofPAMAM-COOH dendrimer. Three samples were used for each group. Nitrite inculture medium was measured using Griess reagent system. *P<0.05,**P<0.01 vs. group of LPS.

FIG. 55 Is a TNF-α release assay. BV-2 cells (passage 16) werestimulated with 100 ng/mL of LPS for 24 hours and 72 hours after 3 hourspre-treatment with the indicated concentration of NAC andPAMAM-(COOH)₄₆-(NAC)₁₈ conjugate. Three samples were used for eachgroup. TNF-α in culture medium was measured using mouse TNF-α ELISA Kit.*P<0.05, **P<0.01 vs. group of LPS; ♦♦P<0.01 vs. group of NAC in sameconcentration.

FIG. 56 shows the effect of dendrimer on TNF-α release. BV-2 cells(passage 16) were stimulated with 100 ng/mL of LPS for 24 hours and 72hours after 3 hours pre-treatment with the chosen concentration (in mM)of PAMAM-COOH dendrimer. Three samples were used for each group. TNF-αin culture medium was measured using mouse TNF-α ELISA Kit. *P<0.05,**P<0.01 vs. group of LPS.

FIG. 57 shows the synthesis of PAMAM-S—S-NAC (1).

FIG. 58 shows the percent total NAC released at pH 7.4 at various GSHconcentrations shown in the graph legend.

FIG. 59 shows the NAC release mechanism of PAMAM-S—S-NAC in the presenceof excess GSH.

FIG. 60 shows a NAC release profile at pH=5 and various GSHconcentrations shown in the graph legend.

FIG. 61 depicts a percent total NAC released at pH 7.4 at various Cysconcentrations shown in the graph legend.

FIG. 62 depicts a percent total NAC released at pH 5 at various Cysconcentrations shown in the graph legend.

FIG. 63 shows a ROS assay, percent reduction in H₂O₂ levels with 24hours NAC, PAMAM-S—S-NAC or dendrimer treatment with simultaneousinduction by LPS stimulation. 100% reduction denotes H₂O₂ concentrationof cells with no induction by LPS (control group). The amount of ROSreleased into the media was measured using Amplex Red. Data are mean±SDof three samples per group, and assessed by t-test. For the freedendrimer, equivalent concentrations of the dendrimer that correspond tothe amount present in the conjugate at the given NAC concentration areshown in bracket.

FIG. 64 shows a ROS assay, percent reduction in H₂O₂ levels with 3 hoursNAC, PAMAM-S—S-NAC or dendrimer treatment followed by LPS stimulation.100% reduction denotes H₂O₂ concentration of cells with no induction byLPS (control group). The amount of ROS released into the media wasmeasured using Amplex Red. Data are mean±SD of three samples per group,and assessed by t-test. The solid bars are the efficacy data for 24hours, whereas the patterned bars denote the response after 72 hours.For free dendrimers, equivalent concentrations of the dendrimers thatcorrespond to the amount present in the conjugate at the given NACconcentration are shown in brackets.

FIG. 65 shows a MALDI-TOF analysis of modified PAMAM dendrimers todetermine the average number of coupled Ethyl-S—S-NAC groups. PAMAM-NH₂before (A) and after (B) reaction with SPDP and followed by NACreaction.

FIG. 66 shows an RP-HPLC analysis of the derivatization of PAMAM-NH2dendrimer with the heterobifunctional cross-linker SPDP and followed byNAC reaction. PAMAM-NH₂ (A); PAMAM-NH—CO-Ethyl-S—S-NAC (B).

FIG. 67 shows a MALDI-TOF analysis of modified PAMAM-COOH dendrimers todetermine the average number of coupled GS—S-NAC groups. PAMAM-COOH (A)and PAMAM-CO-GS-S-NAC (B).

FIG. 68 shows an RP-HPLC analysis of the derivatization of PAMAMdendrimer with the GS-S-NAC. PAMAM-COOH (A), PAMAM-CO-GS-S-NAC (B).

FIG. 69. RP-HPLC UV absorbance chromatograms at 210 nm (arbitrary AUunits) (a) NAC; (b) NAC and NAC-NAC; (c) GSH; (d) GSH and GSSG; (e) GSH,GSSG, NAC, and GS-S-NAC.

FIG. 70 depicts the release of NAC and GS-S-NAC from conjugates (in PBSwith 10 mM GSH).

FIG. 71 shows an efficacy assay of dendrimer-NAC conjugates. BV-2 cellswere treated with 100 ng/mL of LPS and the indicated concentration ofNAC, PAMAM-NH—CO-Ethyl-S—S-NAC (abbreviated as A) conjugate andPAMAM-CO-GS-S-NAC (abbreviated as B) conjugate for 3 hours, and thenincubated with 100 ng/mL of LPS for 24 and 72 hours. Nitrite in culturemedium was measured using Griess reagent system. Data are mean (SD ofthree samples per group, and assessed by t test.

FIG. 72 is an HPLC chromatogram that shows the FITC-G4-OH (1) conjugate.The FITC-G4-OH (1) conjugate showed a single peak at 17.5 in the HPLCchromatogram indicating absence of free FITC using florescent detector(λex=495 nm, λem=521 nm).

FIG. 73 is an ¹H-NMR spectrum of FITC-G4-OH conjugate in DMSO-d6.FITC-G4-OH conjugate was obtained by the reaction of FITC with G4-OH.The integration ratio for FITC and dendrimer corresponds to 2 moleculesof FITC per dendrimer.

FIG. 74 shows the biodistribution of FITC-G4-OH in the brain afterinjection into the subarachnoid space in postnatal day 1 endotoxin andcontrol pups. Increased uptake of FITC-G4-OH was seen in theperiventricular regions in the endotoxin kits (top panel), and with noobvious uptake in the controls (bottom panel) at 24 hourspost-injection, scale bar is 400 μm for lateral ventricle (LV) and forcorpus callosum (CC) the scale bar is 400 μm.

FIG. 75A shows the lectin staining of microglia for cellulardistribution of FITC-G4-OH in the brain following the subarachnoidinjection in postnatal day 1 CP pups. Images show uptake of FITC-G4-OH(Green) in activated microglial cells (Red, Texas-red tagged lectinstaining for microglia), seen as co-localization of staining in cells(arrow) around the lateral ventricle & in the corpus callosum of thenewborn rabbit brain 24 hours post-injection. DAPI staining of nuclei isseen in left hand side of each panel, scale bar is 400 μm for lateralventricle (LV) (top panel); 100 μm (middle panel); 5 μm (bottom panel).

FIG. 75B shows the cellular distribution of FITC-G4-OH in the brainfollowing subarachnoid injection in healthy pups (Lectin staining formicroglia). Images show a few microglial cells (Red, Texas-red taggedlectin staining for microglia) in healthy animals that co-localize withgreen FITC-G4-OH (indicated by arrows), in the periventricular region ofthe newborn rabbit brain at 24 hours post-injection. Nuclei areidentified by DAPI staining. Scale bar is 200 μm for lateral ventricle(LV) (top panel); 50 μm (middle panel) 20 μm (bottom panel).

FIG. 76A shows the cellular distribution of FITC-G4-OH in the brainfollowing subarachnoid injection in CP pups (GFAP staining forastrocytes cells). Images show significant uptake of FITC-G4-OH (Green)in activated astrocytes (Red, Rhodamine labeled GFAP staining forastrocytes), seen as co-localization of staining in the periventricularregion of the newborn rabbit brain at 24 hours post-injection. Nucleiare stained blue with DAPI. Arrow indicates FITC-G4-OH co-localizingwith GFAP staining in activated astrocytes. Scale bar: 20 μm.

FIG. 76B shows the cellular distribution of FITC-G4-OH in the brainfollowing subarachnoid injection in healthy pups (GFAP staining forastrocytes cells). Images show no co-localization of FITC-G4-OH (Green)with astrocytes (Red, Rhodamine labeled GFAP staining for astrocytes) 24hours after subdural injection. The astrocytes are thinner and are notactivated in the healthy animals. A few microglial cells appear to takeup the FITC-G4-OH in the normal newborn rabbit. DAPI is staining nuclei.Scale bar: 50 μm.

FIG. 77 show the cellular distribution of FITC-G4-OH in the brainfollowing subarachnoid injection in postnatal day 1 control kits (MBPstaining for oligodendrocytes cells). Images show no co-localization ofFITC-G4OH(Green) in oligodendrocytes (Red, MBP staining foroligodendrocytes), DAPI for nuclear staining. Scale bar: 100 μm (toppanel); 50 μm (middle panel) 20 μm (bottom panel). Arrow indicatesoligodendrocytes.

FIGS. 78A and B are images following subarachnoid injection of free FITCin the newborn rabbit. Equivalent amount of free FITC was injected andthe animal was euthanized after 24 hours. Astrocytes are stained withrhodamine labeled GFAP (red). Non-specific background staining is notedthroughout the tissue. No co-localization of FITC is seen withastrocytes (A). GFAP and DAPI staining in B; Free-FITC & DAPI stainingin C. DAPI staining of nuclei seen in all slides. Scale bar is 200 μm.

FIG. 79 is a schematic representation of dendrimer nanodevice injectionand biodistribution of FITC-G4-OH in activated microglial and astrocytesits co-localization in the process occurred in cerebral palsy rabbitmodel.

FIG. 80 depict bacterial growth inhibition assays. E. coli was treatedwith the indicated concentration of G₄-PAMAM-NH₂ (A) and (B),G₄-PAMAM-OH (C) and (D), G_(3.5)-PAMAM-COOH (E) and (F) dendrimers for18 hours. The initial concentration used for bacterial seeding was 5×10⁵CFU/mL. Three samples were in each group. Bacterial growth was measuredby turbidity as the optical density at 650 nm using a microplate reader.*P<0.05, **P<0.01, ***P<0.001 VS Positive control.

FIGS. 81A-D are SEM images of E. coli. (A) untreated E. coli (B) 8 hourstreatment of G_(3.5)-PAMAM-COOH (C) 8 hours treatment of G₄-PAMAM-OH (D)8 hours treatment of G₄-PAMAM-NH₂. Magnification 20000×. Scale barsindicate 5 μm. The treatment with dendrimers shows the damage to thebacterial cell wall.

FIG. 82 show the release of intracellular components of E. colisuspensions treated with (A) G_(3.5)-PAMAM-COOH, (B) G₄-PAMAM-OH and (C)G₄-PAMAM-NH₂. Four samples were evaluated in each group. The increase inthe absorbance is an indicator of the compromised cell integrityresulting in leaching of the nuclear components which are absorbed at260 nm.

FIG. 83 show the uptake of NPN by E. coli suspensions treated with (A)G_(3.5)-PAMAM-COOH, (B) G₄-PAMAM-OH and (C) G₄-PAMAM-NH₂. Four sampleswere in each group.

FIG. 84 show the release of cytoplasmic β-galactosidase of E. colitreated with (A) G_(3.5)-PAMAM-COOH, (B) G₄-PAMAM-OH and (C)G₄-PAMAM-NH₂. Four samples were in each group.

FIG. 85 depicts a cytotoxicity assay. (A) Human cervical epithelialEnd1/E6E7 cells and (B) mouse microglial cells were treated with theG₄-PAMAM-OH, G3.5-PAMAM-COOH and G₄-PAMAM-NH₂ dendrimers atconcentrations indicated for MIC values. Three samples were in eachgroup. Cell viability was assessed by MTT method. The proportion ofviable cells in the treated group was compared to that of negativecontrol.

FIG. 86 show the flow cytometry of the cell entry dynamics of (A)G₄-PAMAM-OH-FITC in E. coli and (B) BV-2 microglial cell line. The logof FITC absorption intensity (FL1-H on x-axis) is plotted against thenumber of cells (counts on y-axis). The maximum uptake ofG₄-PAMAM-OH-FITC in E. coli occurs at 3 hours. The rapid cellular uptakeof G4-PAMAM-OH-FITC within 15 minutes in microglial cells is evident.The transport of conjugate into microglial cell increased withincreasing time. Confocal microscopy images (400×) showed thatG₄-PAMAM-OH-FITC appeared to be mainly localized in the cytoplasm ofBV-2 cells while the nucleus appeared to be relatively free of thepresence of any fluorescence at this time point (C).

FIG. 87 show the placental tissue (0.3 g) was homogenized in 1 ml RIPAlysis buffer. The homogenate was kept on ice for 30 minutes and theprotein concentration of supernatant was determined. Cytokinesconcentrations were measured in the total protein fraction using ELISA.*P<0.05, ***P<0.001 VS Normal control. ▴▴P<0.01, ▴▴▴P<0.001 VS E.coligroup.

FIG. 88 are MALDI TOF MS spectra for G4-PAMAM-O-GABA-Boc (5) (Mw=15,960Da), G4-PAMAM-O-GABA-NH₂ (6) (Mw=14,949 Da), G4-PAMAM-O-GABA-NH-FITC (1)(Mw=15,805 Da) and G4-PAMAM-O-GABA-NH-Alexa (2) (Mw=16065 Da) showingthe corresponding mass.

FIG. 89 are HPLC chromatograms for G4-PAMAM-O-GABA-NH₂ (6) (UV channel),G4-PAMAM-O-GABA-NH-FITC (1) (Fluorescent channel) andG4-PAMAM-O-GABA-NH-Alexa 488 (2) (Fluorescent channel). The retentiontime of G4-PAMAM-O-GABA-NH₂ is 16.2 minutes and the FITC and Alexatagged G4-PAMAM-O-GABA-NH₂ show a peak appearing at 17.5 and 15.5minutes respectively.

FIGS. 90 A and B show transport across membranes. (A) The transport ofG₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated) across thefetal membrane comprising amnion and chorion together over 30 hours inthe side by side diffusion chamber. (B) The FITC shows a rapid transportacross the membrane in 5 hours (˜20%), while the dendrimers shownegligible transport of ≤3% in 5 hours. The concentrations of D-FITCstudied were 0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3mg/mL.

FIGS. 91A and B show transport across membranes. (FIG. 91A) Thetransport of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated)across the chorion stripped off fetal membrane. The amnion was placed inthe side by side diffusion chamber over 30 h to study the transport ofdendrimers. (FIG. 91B) The FITC shows a rapid transport across themembrane (50%) in 5 hours, while the dendrimers show negligibletransport of ≤3% in 5 hours. The concentrations of D-FITC studied were0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3 mg/mL.

FIGS. 92A and B show transport across membranes. (FIG. 92A) Thetransport of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) and FITC (unconjugated)across the amnion stripped off fetal membrane. The chorion was placed inthe side by side diffusion chamber over 30 hours to study the transportof dendrimers. (FIG. 92B) The FITC shows a rapid transport across themembrane (˜25%) in 5 hours, while the dendrimers show negligibletransport of ≤3% in 5 hour. The concentrations of D-FITC studied were0.6 mg/mL and 3 mg/mL. The concentration of FITC was 0.3 mg/mL.

FIG. 93 depicts the Permeability coefficient for dendrimer measuredacross the (A) chorioamnion (B) amnion and (C) chorion. Theconcentrations of G₄-PAMAM-O-GABA-NH-FITC (D-FITC) studied were 0.6mg/mL and 3 mg/mL. The permeability coefficient of 0.6 mg/mL and 3 mg/mLD-FITC through (A) chorioamnion was 7.5×10⁻⁸ and 5.8×10⁻⁸ respectively(B) amnion was 1.86×10⁻⁸ and 2.08×10⁻⁷ and (C) chorion was 2.94×10⁻⁸ and2.94×10⁻⁸ cm²/s

FIG. 94 depicts the permeability coefficient for FITC (unconjugated)measured across the chorioamnion, amnion and chorion. The concentrationof FITC was 0.3 mg/mL. The permeability coefficient of FITC throughchorioamnion was 7.93×10⁻⁷, amnion was 2.26×10⁻⁶ and chorion was1.32×10⁻⁶ cm²/s.

FIG. 95A shows the H and E stained human chorioamniotic (fetal)membrane. AE=amniotic epithelium, AM=amniotic mesoderm, CM=chorionicmesoderm, CT=chorionic trophoblast, DE=decidua comprising the stromalcells. For the transmembrane study the amniotic epithelium was placedfacing the receptor cell to study the transport of dendrimer frommaternal side (extra-amniotic cavity) to the fetal side.

FIG. 95B shows the human chorioamniotic (fetal) membrane showing thenuclei stained blue with DAPI (control membrane without the treatmentwith G₄-PAMAM-O-GABA-NH-Alexa (D-alexa) (20×). The negative controlsrabbit isotype and mouse isotype replaced the primary antibodies.AE=amniotic epithelium, CAM=chorioamniotic mesoderm, CT=chorionictrophoblast, DE=decidua comprising the stromal cells. For thetransmembrane study the amniotic epithelium was placed facing thereceptor cell to study the transport of dendrimer from maternal side(extra-amniotic cavity) to the fetal side.

FIG. 96 shows the transmembrane transport of G₄-PAMAM-O-GABA-NH-Alexa(D-alexa) across the human fetal membrane at different time points (30minutes, 1, 2, 2.5, 3, 3.5 and 4 hours respectively) (20×). The nucleiare stained as blue (DAPI), the trophoblast cells in the chorion regionare stained cytokeratin positive (red) and the stromal cells in thedecidua are stained vimentin positive (magenta). The D-alexa (green) canbe seen advancing through the different regions (the different regionsare marked in the control membrane shown in bottom panel). At initialtime points (30 min to 2 hours) the dendrimer is seen in mostly in thedecidua and stromal cells and at time points 3 to 4 hours the dendrimersseem to diffuse into the chorionic trophoblast region. The image withoutcytokeratin and vimentin shows the diffusion of dendrimer throughout thedecidua and trophoblast cells (4 hours, bottom panel, center).AE=amniotic epithelium, CAM=chorioamniotic mesoderm, CT=chorionictrophoblast, DE=decidua comprising the stromal cells. For thetransmembrane study the amniotic epithelium was placed facing thereceptor cell to study the transport of dendrimer from maternal side(extra-amniotic cavity) to the fetal side.

FIG. 97a shows colocalization images for the G₄-PAMAM-O-GABA-NH-Alexa(D-alexa) in the decidual stromal cells at 4 hours. The stromal cellsare vimentin positive (magenta) and the nuclei of all the cells arestained blue. The D-alexa is seen in green. The internalization ofD-alexa in the nuclei and cytoplasm of stromal cells can be seen fromthe merged composite image. The colocalized D-alexa with nuclei appearsas cyan (63×). The arrows identify the cells showing cellular uptake ofdendrimer (63×). Also the dendrimer seems largely in the interstitialregions.

FIG. 97b shows colocalization images for the G₄-PAMAM-O-GABA-NH-Alexa(D-alexa) in the chorionic trophoblast region at 4 hours. The chorionictrophoblast cells are cytokeratin positive (red) and the nuclei of allthe cells is stained blue. The D-alexa is seen in green. Theinternalization of D-alexa in the nuclei of trophoblast cells is seenfrom the merged composite image. The arrows identify the cells showingcellular uptake of dendrimer. The colocalized D-alexa with nucleiappears as cyan (63×). Also the bottom panel shows that dendrimer islargely in the interstitial regions (20×).

FIG. 98 shows the dendrimer biodistribution in the brain, whereinanimals are sacrificed at 6 or 24 hours and brain sections are stainedwith Rhodamine labeled GFAP or tomato lectin for co-localization.

FIG. 99 shows a scheme of the PAMAM-NH—CO—Pr—S—S-NAC conjugate taggedwith fluorescent dye FITC for a cell uptake study.

FIG. 100 shows a scheme of S-(2-Thiopyridyl) glutathione prepared fromthe reaction of 2,2′-dithiodipyridine in excess and the correspondingpeptide in a mixture of methanol and water at room temperature.

FIG. 101 shows a scheme of the carboxylic acid group of FITC, conjugatedwith —OH groups of PG4-OH dendrimer using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as acoupling agent.

FIG. 102 shows a schematic representation for the synthesis ofN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

FIG. 103 shows a schematic representation for the synthesis ofPAMAM-NH—CO—Pr—S—S-NAC.

FIG. 104 shows a schematic representation for the synthesis offluorescently labeled G₄-PAMAM-dendrimers; G4-PAMAM-O-GABA-NH-FITC (1)and G4-PAMAM-O-GABA-NH-Alexa (2).

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention provides nanodevices formed oftherapeutically active agents or compounds (hereinafter “agent”)conjugated or attached to a dendrimer or multiarm PEG. The attachmentcan occur via an appropriate spacer that provides a disulfide bridgebetween the agent and the dendrimer. The nanodevices are capable ofrapid release of the agent in vivo by thiol exchange reactions under thereduced conditions found in body. The dendrimers disclosed herein caninclude, but are not limited to, PAMAM dendrimers. The embodimentsdisclosed herein are not limited to this class, and other types ofdendrimer such as polyester or PPI can be used. The multiarm PEG polymercomprises polyethylene glycol having 2 and more branches bearingsulfhydryl or thiopyridine terminal groups; however, embodimentsdisclosed herein are not limited to this class and PEG polymers bearingother terminal groups such as succinimidyl or maleimide terminations canbe used. The PEG polymers in the molecular weight 10 kDa to 80 kDa canbe used.

The term “nanodevices” as used herein it intended to be defined as acombination of a dendrimer with a therapeutically active agent. Thesenanodevices include an agent that is attached or conjugated to PAMAMdendrimers or multiarm PEG, which are capable of preferentiallyreleasing the drug intracellularly under the reduced conditions found invivo. In other words, the nanodevice is a dendrimer linked to an activemolecule. The nanodevices, when administered by i.v. injection, canpreferentially cross the blood brain barrier (BBB) only under diseasedcondition and not under normal conditions. The nanodevices can also beuseful for targeted delivery of the therapeutics in neuro-inflammation,cerebral palsy, ALS and other CNS diseases.

The nanodevices can be administered via parenteral, topical and oralroute either by itself or as a part of a formulation such as hydrogels,nanoparticle or microparticles, suspensions, gels, ointments, powders,tablets, capsules and solutions. The nanodevice composition can beadministered parenterally by subdural, intravenous, intra-amniotic,intraperitonial, subcutaneous routes, topically on skin, eye and othermucosal membranes such as vaginal, orally either as solid or liquiddosage form. Further, the nanodevice can be formed for oral or topicalapplication wherein the composition is administered in form of solution,suspension, powder, tablet or capsule for oral administration and asgel, ointment, solution or as a patch for topical administration. Thenanodevice is capable of targeting and or rapidly releasing ordelivering the therapeutically active agent at the site of action orabsorption either intracellularly or in interstitial spaces.

The term “dendrimer” as used herein is intended to include, but is notlimited to, a molecular architecture with an interior core, interiorlayers (or “generations”) of repeating units regularly attached to thisinitiator core, and an exterior surface of terminal groups attached tothe outermost generation. Examples of dendrimers include, but are notlimited to, PAMAM, polyester, polylysine, and PPI. The PAMAM dendrimerscan have carboxylic, amine and hydroxyl terminations and can be anygeneration of dendrimers including, but not limited to, generation 1PAMAM dendrimers, generation 2 PAMAM dendrimers, generation 3 PAMAMdendrimers, generation 4 PAMAM dendrimers, generation 5 PAMAMdendrimers, generation 6 PAMAM dendrimers, generation 7 PAMAMdendrimers, generation 8 PAMAM dendrimers, generation 9 PAMAMdendrimers, or generation 10 PAMAM dendrimers. Dendrimers suitable foruse with the present invention include, but are not limited to,polyamidoamine (PAMAM), polypropylamine (POPAM), polyethylenimine,polylysine, polyester, iptycene, aliphatic poly(ether), and/or aromaticpolyether dendrimers. Each dendrimer of the dendrimer complex may be ofsimilar or different chemical nature than the other dendrimers (e.g.,the first dendrimer may include a PAMAM dendrimer, while the seconddendrimer may comprises a POPAM dendrimer). In some embodiments, thefirst or second dendrimer may further include an additional agent. Themultiarm PEG polymer includes a polyethylene glycol having at least twobranches bearing sulfhydryl or thiopyridine terminal groups; however,embodiments disclosed herein are not limited to this class and PEGpolymers bearing other terminal groups such as succinimidyl or maleimideterminations can be used. The PEG polymers in the molecular weight 10kDa to 80 kDa can be used.

In another embodiment of the present invention, the dendrimer complexcan include multiple dendrimers. For example, the nanodevice can includea third dendrimer; wherein the third-dendrimer is complexed with atleast one other dendrimer. Further, a third agent can be complexed withthe third dendrimer. In another embodiment, the first and seconddendrimers are each complexed to a third dendrimer, wherein the firstand second dendrimers are PAMAM dendrimers and the third dendrimer is aPOPAM dendrimer. Additional dendrimers can be incorporated withoutdeparting from the spirit of the invention. When multiple dendrimers areutilized, multiple agents can also be incorporated. The presentinvention is not limited by the number of dendrimers complexed to oneanother.

The term “spacers” as used herein is intended to include compositionsused for linking a therapeutically active agent to the dendrimer. Thespacer can be either a single chemical entity or two or more chemicalentities linked together to bridge the polymer and the therapeutic agentor imaging agent. The spacers can include any small chemical entity,peptide or polymers having sulfydryl, thiopyridine, succinimidyl,maleimide, vinylsulfone, and carbonate terminations.

The spacer can be chosen from among a class of compounds terminating insulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone andcarbonate group. The spacer can comprise thiopyridine terminatedcompounds such as dithiodipyridine, N-Succinimidyl3-(2-pyridyldithio)-propionate (SPDP), Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or Sulfo-LC-SPDP.The spacer can also include peptides wherein the peptides are linear orcyclic essentially having sulfhydryl groups such as glutathione,homocysteine, cysteine and its derivatives, arg-gly-asp-cys (RGDC) (SEQID NO: 4), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDFC)) (SEQ ID NO: 5),cyclo(Arg-Gly-Asp-D-Tyr-Cys) (SEQ ID NO: 6),cyclo(Arg-Ala-Asp-d-Tyr-Cys) (SEQ ID NO: 7). The spacer can be amercapto acid derivative such as 3 mercapto propionic acid, mercaptoacetic acid, 4 mercapto butyric acid, thiolan-2-one, 6 mercaptohexanoicacid, 5 mercapto valeric acid and other mercapto derivatives such as 2mercaptoethanol and 2 mercaptoethylamine. The spacer can bethiosalicylic acid and its derivatives,(4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene,(3-[2-pyridithio]propionyl hydrazide, The spacer can have maleimideterminations wherein the spacer comprises polymer or small chemicalentity such as bis-maleimido diethylene glycol and bis-maleimidotriethylene glycol, Bis-Maleimidoethane, bismaleimidohexane. The spacercan comprise vinylsulfone such as 1,6-Hexane-bis-vinylsulfone. Thespacer can comprise thioglycosides such as thioglucose. The spacer canbe reduced proteins such as bovine serum albumin and human serumalbumin, any thiol terminated compound capable of forming disulfidebonds. The spacer can include polyethylene glycol having maleimide,succinimidyl and thiol terminations.

The term “therapeutically active agents” or “bioactive compounds” asused herein is intended to include antibiotics, antioxidants, steroids,NSAIDs, progesterone, and thalidomide. The therapeutic agent can alsoinclude a drug or modified form of drug such as prodrugs and analogs.The therapeutic agent can also be siRNAs, microRNAs, DNA, RNA, andpeptide drugs. Other examples of agents include, but are not limited to,β-lactum, tetracycline and microlides antibiotics, wherein the β-lactumantibiotics comprise penicillins such as ampicillin, cephalosporinsselected in turn from the group consisting of cefuroxime, cefaclor,cephalexin, cephadroxil and cepfodoxime proxetil the tetracyclineantibiotics comprise doxycycline and minocycline, the microlideantibiotics comprise azithromycin, erythromycin, rapamycin andclarithromycin, fluroquinolones selected in turn from the groupconsisting of ciprofloxacin, enrofloxacin, ofloxacin, gatifloxacin,levofloxacin and norfloxacin, an antioxidant drug comprisesN-acetylcysteine. An anti-inflammatory drug can be a nonsteroidal drugsuch as indomethacin, aspirin, acetaminophen, diclofenac sodium andibuprofen; the steroidal anti-inflammatory drug can be dexamethasone.The corticosteroids can be fluocinolone acetonide andmethylprednisolone. The peptide drug can be streptidokinase. Thetherapeutic agent can be a PAMAM dendrimer with amine or hydroxylterminations. The targeting moiety can be folic acid, RGD peptideseither linear or cyclic, TAT peptides, LHRH and BH3.

More specifically, the nanodevices linked to a bioactive compound ortherapeutically active agent, examples of which are disclosed inembodiments, can be used to perform several functions includingtargeting, localization at a diseased site, releasing the drug, andimaging purposes. The nanodevice linked to the bioactive compounds ortherapeutically active agents can be used in therapies. For example, thenanodevices of the present invention can incorporate agents and/orimaging agents with the dendrimers or multiarm PEG polymer. Thenanodevices can be tagged with or without targeting moieties such that adisulfide bond between the dendrimer and the agent or imaging agent isformed via a spacer or linker molecule. The nanodevices disclosed hereincan rapidly release the agent by the cleavage of the disulfide bondin-vivo. For example, G4 PAMAM-NAC, as disclosed herein, can beadministered to a patient for treatment of inflammation associated withmaternal fetal infections involving neuro-inflammation associated withcerebral palsy. Because of the site specific delivery, less of the agenthas to be administered. This has no impact on the bioavailability of theagent, and in fact the agent is delivered via the nanodevice is ten to ahundred times more efficacious than the free drug. The enhancedbioavailability of the agent is due to the inhibition of the plasmaprotein binding and enhancement of the intracellular delivery. Inconjunction with the decreased amount of agent being administered, thereare fewer side effects without a corresponding decrease in efficacy. Thedisclosed nanodevices deliver agents having a higher efficacy than thedrug itself. The nanodevices comprising PAMAM dendrimer or PEG linked toseveral drugs by disulfide linkage offers the advantage of rapid drugrelease, site specific delivery of drugs. Unlike drugs linked by amideor ester bonds where the hydrolysis takes place slowly, the disulfidelinkages deliver the drug rapidly. As shown in the Examples, thecompounds are ten to a hundred times more efficacious than the freedrug.

The nanodevices of the present invention have selective permeabilities.For example, the nanaodevices do not cross the placenta and the amnioticmembranes such that on injection into the amnion or intra-amnioticfluids the nanodevices exhibit no or minimal leaching into the tissuesand vasculature of the pregnant woman, restricting the exposure to thebaby or fetus. Alternatively, the nanodevice, including a dendrimerlinked to a bioactive compound or therapeutically active agent, can beused to treat the pregnant woman, thereby restricting the exposure ofthe nanodevices into the fetus or the conceptus.

In light of the selective permeability, the nanodevice, a dendrimerlinked to a bioactive compound or therapeutically active agent, can beused for treating maternal fetal infections such as chorioamnionitis orbacterial vaginosis or any other ascending genital infection, urinarytract infections, HIV/AIDS, herpes, Group B streptococcus andlisteriosis. Specifically, the nanodevice includes a polymer, and atherapeutically active agent. Alternatively, an imaging agent and/ortargeting moiety can also be incorporated. The therapeutically activeagent, or imaging agent, and/or targeting moiety can be eithercovalently attached or intra-molecularly dispersed or encapsulatedwithin the dendrimer. The attachment occurs via one or more spacermolecules. The spacer molecules, as disclosed above, can end indisulfide, ester or amide bonds. The nanodevice is administered eitherin form of injectable solution or suspension or topically in form of apatch, gel, ointment or solution.

Additionally, the nanodevice composition, including a dendrimer linkedto a bioactive compound or therapeutically active agent, can alsoselectively cross the blood-brain barrier. Thus, the nanodevices of thepresent invention can be used to administer an agent to the brain of apatient. The nanodevice only crosses the blood-brain barrier inappreciable amounts when diseased conditions of the central nervoussystem especially in neuroinflammatory conditions such as white matterinjury and cerebral palsy and does not cross the blood brain barrier innormal conditions. The nanodevices can therefore be used to selectivelyadminister agents to brain tissues while limiting the side effects ofthe agents.

The nanodevice composition, including a dendrimer linked to a bioactivecompound or therapeutically active agent, can also selectively targetmicroglia and astrocytes. Thus, the nanodevices of the present inventioncan be used to target and treat neuroinflammation. After the nanodeviceslocalize at the microglia and astrocytes, which play a key role in thepathogenesis of several neurodegenerative diseases, including cerebralpalsy. The agent that is incorporated into the nanodevice can deliverthe agent to and near the site of localization This enables thenanodevice to be used to locate and treat inflammation.

A specific nanodevice for treating maternal fetal infections can includea dendrimer or multiarm PEG polymer and a therapeutically active agent.Alternatively, an imaging agent and/or targeting moiety can also beincluded. The therapeutically active agent, imaging agent, and/ortargeting moiety can be either covalently attached or intra-molecularlydispersed or encapsulated. The dendrimer is preferably a PAMAM dendrimerup to generation 10, having carboxylic, hydroxyl, or amine terminations.The PEG polymer is a star shaped polymer having 2 or more arms and amolecular weight of 10 kDa to 80 kDa. The PEG polymer has hassulfhydryl, thiopyridine, succinimidyl, or maleimide terminations. Thedendrimer is linked to the targeting moiety, imaging agents, and/ortherapeutic agents via a spacer ending in disulfide, ester or amidebonds.

The nanodevice can also be used for intrauterine administration. Forsuch uses, the nanodevice is administered in the form of an injectablesolution, hydrogel or suspension directly into the uterus, and includesa dendrimer or multiarm PEG polymer and a therapeutically active agent.

In a specific embodiment, the nanodevice is based on PAMAM dendrimers ormultiarm PEG polymers linked to drugs by disulfide linkages viaappropriate spacer or linker molecules. These G4 PAMAM-drug or PEG-drugconjugates can be used as funtionalized nanocarriers or nanodevicescapable of rapid release of the drugs at the target site, ensuring thebioavailability of the drugs. One suitable linker between the drug andthe dendrimer or multiarm PEG polymer is a disulfide linker, tofacilitate the cleavage of the drug into active form in the presence ofreducing agents such as glutathione, a chemical entity found in thehuman body.

These nanodevices based on the PAMAM dendrimers or multiarm PEG polymerlinked to various drugs, targeting moieties, imaging agent by disulfidelinkages offer several advantages: (1) the composition in itself acts asa device capable of targeting, localizing and releasing the drug; (2)the drugs are only released in the redox environment usually found ininfected tissues or cells such as tumor, inflammation associated withseveral infections; (3) the composition can preferentially deliver thedrug only to the mother in pregnant woman sparing the baby and conceptusand vice versa, for example to treat the fetus or conceptus withoutaffecting the pregnant woman; and (4) the nanodevices can be formulatedin new dosage forms including tablets, injections, gels powderscapsules, films, etc. Since, PEGs are approved for human use there is anadditional benefit to using the nanodevices of the present invention.

Thus, the nanodevices of the present invention can be used to treatdiseases related to chronic inflammation. Examples of such diseasesinclude, but are not limited to, heart attack, Alzheimer's disease,congestive heart failure, stroke, arthritis, aortic valve stenosis,kidney failure, lupus, asthma, psoriasis, pancreatitis, allergies,fibrosis, surgical complications, anemia, fibromyalgia, and otherinflammatory diseases including, but not limited to, neuroinflammation.The nanodevices can also be used as antibacterial and/or antimicrobialdevices.

By way of example, NAC is a drug very extensively investigated andstudied. It is also investigated for neuro-inflammation associated inmaternal fetal infections. However, NAC suffers from low bioavailabilitydue to high plasma protein binding. The nanodevice compositionsdisclosed herein are designed to overcome the plasma protein bindingwithout affecting the activity of NAC.

In fact, G4 PAMAM-NAC can be ten to a hundred times more efficacious invivo than the free drug NAC by single i.v. administration. The free drugNAC exhibits very high plasma protein binding resulting in reducedbioavailability. One of the major advantages of this nanodevice is thatit enhances the bioavailability by restricting the unwanted drug plasmaprotein interactions and selectively results in rapid release of thedrug intracellularly to exhibit the desired therapeutic action. Theenhanced efficacy of the nanodevices without any significant toxicity invitro and in vivo is exemplified in the embodiments disclosed herein.

The high payload of the drug NAC in the G4 PAMAM-NAC requires very smallquantities (over 10 mg) of the carrier, PAMAM dendrimer, therebyreducing the amounts administered daily. A decreased quantity of agentlimits the side effects associated with the agent. Since thebioavailability of the agent remains high, the positive effects of theagent are not lowered despite the administration of smaller quantitiesof agent.

The nanodevices including the dendrimer-drug conjugates, restricts itsbiodistribution to tissues and organ and preferentially deliver the drugat the target site thereby reducing the undesired side effects.

Dendrimer nanodevices effectively transport across the BBB, and offer anew method for targeted drug delivery in brain injuries. The resultsdisclosed herein demonstrate that G4-PAMAM-S—S-NAC conjugates can beused to specifically target activated microglial cells and astrocytes inneuroinflammatory disorders. The therapeutic efficacy ofG4-PAMAM-S—S-NAC dendrimer conjugate was evaluated after two days ofanimal treatment with lipopolysaccharide (LPS) to induce white matterinjury and hypomyelination in the developing rabbit brain (an animalmodel of Cerebral Palsy).

NAC selectively delivered from the G4-PAMAM-S—S-NAC nanodevices stronglysuppressed pro-inflammatory cytokines (TNF-α, IL-6 mRNA), inflammatorysignaling factors, including NFκB and nitrotyrosine, and enhanced GSHlevel. The G4-PAMAM-S—S-NAC was found to be ten to a hundred times moreefficacious compared with free NAC. This supports a conclusion that theG4-PAMAM-S—S-NAC traversed across the BBB. The targeted delivery of NACfrom dendrimer nanodevice to actived microglial cells improved the motordeficits and attenuated recovery from the LPS-induced brain injury in aneonatal rabbit model of cerebral palsy.

A significant reduction in proinflammatory cytokines (TNF-α, IL-6 mRNA)was observed on administration of G4-PAMAM-S—S-NAC nanodevices. The kitstreated with NAC and G4-PAMAM-S—S-NAC showed a decrease in fetalinflammation response with improvement of motor deficits when comparedto the kits that were treated with saline. The kits that were treatedwith G4-PAMAM-S—S-NAC conjugates had less behavioral changes and lowermicroglial activation in the brain when compared to the kits thatreceived NAC alone due to the sustained delivery of NAC fromG4-PAMAM-S—S-NAC conjugate. The results indicate that G4-PAMAM-S—S-NACconjugates have a greater effect than NAC alone since it ispreferentially taken up by activated macrophages and microglial cells,reducing the inflammatory and oxidative and nitrosative effects.

Treatment with G4-PAMAM-S—S-NAC nanodevices reduced white matter injuryand microglia activation. A significant reduction in dose of NAC wasobserved when administered as G4-PAMAM-S—S-NAC to elicit the similarresponse as that observed for free NAC. Both free NAC at concentration100 mg/kg and G4-PAMAM-S—S-NAC at concentration 10 mg/kg, 10 mg elicitidentical responses, demonstrating that on conjugating to dendrimer areduction in dose is achieved. G4-PAMAM-S—S-NAC at lower concentrationsthan free NAC shows significant protective effects against LPS-inducedbrain injuries, suppression of TNF-α and down-regulation of IL-6activity. This activity of the dendrimer-NAC conjugates may beattributed to its ability to interfere with the early inflammatoryresponses by blocking or modifying the signal transduction factor NF-κBand nitrotyrosine, thereby modulating cellular activation.

The down-regulation of TNF-α and IL-6 in hippocampus, is likely to beattributed to the preferential biodistribution of dendrimer nonodeviceswith specific cell uptake by microglia cell in the brain. Thedendrimer-NAC nanodevices can be used for treatment of pregnant womendeveloping clinical symptoms associated with maternal infection, withincreased risk of developing PVL and CP in infants. The results showthat inhibition of microglial cells, astrocytes with Dendrimer-NACdecreased the white matter injury in the newborn rabbit brain. Further,the dendrimers exhibit sustained release of conjugated drugs, andenhance the effectiveness of drugs over a prolonged period. At lowerdose, Dendrimer-NAC conjugates were more effective than NAC alone. Thedendrimer-NAC conjugates seem to offer more advantages includingsignificant dose reduction, enhanced bioavailability, and reduction indosing.

As another example, 6 and 8 arm PEG-NAC conjugates released 74% of NACin the intracellular GSH concentration (2 and 10 mM), within 2 hours. Ata concentration range of between 0.008-0.8 mM, the conjugates werenontoxic to the microglial cells. At an equimolar concentration of NAC(0.5 mM) the 6-arm-PEG-S—S-NAC and 8-arm-PEG-S—S-NAC were more efficientin inhibition of GSH depletion than the free NAC. Both 6 and8-arm-PEG-S—S-NAC conjugates, each at 0.5 mM and 5 Mm concentrationshowed significant inhibition in ROS production when compared to freeNAC at equimolar concentrations. The studies demonstrate that theconjugates are superior in inhibition of the NO production as comparedto the free NAC. At the highest concentration (5 mM), the free drugreduced the H₂O₂ levels and nitrite levels by 30-40%, whereas theconjugates reduced the H₂O₂ and nitrite levels by more than 70%. Thisshows that the conjugates are able to traffic the drug inside the cells,and release the drug in the free form and are significantly moreefficacious than the free drug. At 5 mM concentration 6-arm-PEG-S—S-NACconjugate (1) showed significant inhibition (70%) of TNF-α productionwhen compared to equivalent concentration of NAC (Pb0.05).8-arm-PEG-S—S-NAC conjugate (3) showed significant inhibition of TNF-αproduction (70%) at 5 mM when compared to equivalent concentration ofNAC (Pb0.05 and Pb0.01). PEGylated NAC is a nanodevice with utility forthe pharmaceutical industry, as PEGs are approved for human use and thisdevice addresses limitations of NAC and provides greater efficacy.

The Examples below are included to demonstrate preferred embodiments ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the Examples represent techniques andcompositions discovered by the inventors to function well in thepractice of embodiments disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstil obtain a like or similar result without departing from the spiritand scope of embodiments disclosed herein.

EXAMPLES Example 1

PAMAM-NH—CO-Ethyl-S—S-NAC Conjugate

For the preparation of PAMAM-PDP generation 4, SPDP (0.5 equivalent) inethanol (10 mL) was added to a solution of PBS buffer pH 7.4 (20 mL) andPAMAM-NH₂ dendrimer (1 equivalent) to provide sufficient modificationwhilst preventing loss of product due to the precipitation of highlymodified dendrimer. The reaction was stirred at room temperature for 2hours. To this reaction N-Acetyl cysteine was added (1 equivalent) atonce, and the reaction was stirred at room temperature for 4 hours. Thereaction was monitored with HPLC. After completion of reaction, thereaction mixture was diluted with water and lyophilized to get crudeproduct. The solid crude product was diluted with water and dialyzedagainst DMSO followed by PBS (pH=7.4) to remove by-products and theexcess of reactants, and then replaced with deionized water (1 in 41times) for 12 h to remove salts. The water was lyophilized to get pureproduct in good yield (71%).

Example 2

PAMAM-CO-GS-S-NAC Conjugate

Step 1. S-(2-thiopyridyl) glutathione

S-(2-thiopyridyl) glutathione was prepared from the reaction of 2,2¹-dithiodipyridine (2 equivalent) and GSH (1 equivalent) in a mixtureof methanol and water (1:1) stirred for 15 hours at room temperature.Upon completion of the reaction (monitored by TLC), most of methanol wasremoved in vacuo and the residue was dissolved in water washed withdichloromethane. The aqueous solution was subjected to reverse phase(RP) HPLC purification, and lyophilization of the eluent gave the pureproduct as a white solid in 80% yield. Calculated mass: 416. ESI m/z 417(M+H). ¹H-NMR (300 MHz, d₆-DMSO) δ/ppm 1.98-2.11 (2H, m), 2.22-3.02 (2H,m), 2.98-3.08 (1H, m), 3.18-3.22 (1H, m), 3.65-3.71 (2H, m), 3.95-402(1H, m), 4.57-4.62 (1H, m), 7.0-7.07 (1H, m), 7.72-7.87 (2H, m), 8.24(2H, br.s., NH), 8.42-8.48 (3H, m, NH₂, aromatic).

Step 2. N-Acetyl-glutathione

S-(2-thiopyridyl) glutathione (1 equivalent) was dissolved in PBS bufferpH=7.4 (5 mL) and added NAC (1 equivalent) at once, and the reactionmixture was stirred at room temperature for 2 hours. After completion ofthe reaction, dichloromethane was added and the organic layer wasseparated to remove the corresponding thione by-product, this processwas repeated five times. The aqueous solution was subjected to reversephase (RP) HPLC purification, and lyophilization of the eluent gave thepure product as a white solid in 79% yield. Calculated mass: 468. ESIm/z 467 (M−H). ¹H NMR (300 MHz, d₆-DMSO) δ/ppm, 1.81 (3H, s), 1.90-2.0(2H, m), 2.28-2.37 (2H, m), 2.79-2.86 (3H, m), 3.07-3.17 (2H, m),3.69-3.78 (2H, m), 4.40-4.46 (1H, m), 4.52-4.58 (1H, m). ¹³C-NMR (75,MHz d₆-DMSO) δ/ppm, 22.35, 26.09, 30.86, 51.26, 51.32, 51.64, 52.02,169.36, 169.44, 170.89, 171.27, 172.02, 172.07.

Step 3. PAMAM-CO-GS-S-NAC Conjugate

The anionic PAMAM-COOH generation-3.5 dendrimer (1 equivalent) wasdissolved in DMSO/DMF (3:2, 20 mL) and DIEA (1 equivalent) and PyBop (1equivalent) were added and the reaction stirred for 1 hour. To this asolution of N-Acetyl-glutathione (1.5 equivalent) was added in DMSO (10mL) was stirred for 12 hours at room temperature. The reaction wasmonitored with HPLC. After completion of reaction, it was diluted withwater and lyophilized to get crude product. The solid crude product wasdiluted with water and dialyzed against DMSO followed by PBS (pH=7.4) toremove by-products and the excess of reactants and then replaced withdeionized water for 12 hours to remove salts. The water was lyophilizedget pure product in 78% yield. Raghu/Bing Efficacy data for COOHdendrimer were determined.

Example 3

PAMAM-O-GABA-NH—CO-Ethyl-S—S-NAC

Step 1. Synthesis of PAMAM-O-GABA-BOC

A solution of BOC-GABA-OH (1.5 equivalent)) in DMSO/DMF (3:1) was cooledto 0° C. and then treated with a solution of EDC (1.5 equivalent), DMAP(0.01 eq) and G4-OH, PAMAM dendrimer (1 equivalent) in DMSO/DMF (3:1).This was left to stir at room temperature for 24 hours. The reactionmixture was purified on dialysis with DMSO (3 times) to removeby-products and the excess of reactants and after dialysis the solventwas removed under lyophilization to get pure compound.

Step 2. Synthesis of PAMAM-O-GABA-NH₂

To a stirred solution of PAMAM-O-GABA-BOC (1 equivalent) was treatedwith trifloroacetic acid and dichloromethane (1:1, 10 mL). The reactionwas stirred at room temperature for 10 min. After completion of thereaction trifloroacetic acid/dichloromethane was removed underrotavapor. Reaction mixture was neutralized with PBS (pH=7.4) ondialysis with water (3 times) and solvent was removed underlyophilization to get pure compound.

Step 3. PAMAM-O-GABA-NH—CO-Ethyl-S—S-NAC

For the preparation of PAMAM-O-GABA-PDP generation 4, SPDP (1equivalent) in ethanol (10 mL) was added to a solution of PBS buffer pH7.4 (20 mL) and PAMAM-O-GABA-NH₂ dendrimer (1 equivalent) to providesufficient modification whilst preventing loss of product due to theprecipitation of highly modified dendrimer. Reaction was stirred at roomtemperature for 2 hours. To this reaction N-Acetyl cysteine was added (1eq) at once, and stirred the reaction at room for 4 hours. The reactionwas monitored with HPLC. After completion of reaction, the reactionmixture was diluted with water and lyophilized to get crude product. Thesolid crude product was diluted with water and dialyzed against PBS(pH=7.4) to remove by-products and the excess of reactants and thenreplaced with deionized water (3 times) dialyzed for 12 hours to removesalts. The water was lyophilized to get pure product in good yield.

Example 4

In Vivo Evaluation of the Efficacy of G4-PAMAM-S—S-NAC Nanodevices inRabbit Model of Cerebral Palsy

New Zealand White rabbits (CoVance Research Products Inc., Kalamazoo,Mich.) with timed pregnancies confirmed with breeders (having a historyof delivering 7-11 kits per litter) underwent laparotomy under generalanesthesia (2-3% isoflurane by mask) on gestational day 28 (E28, termpregnancy is 31-32 days). 1 mL of saline for the control group (n=6) or1 mL of saline containing 20 μg/kg of LPS (Escherichia coli serotypeO127: B8 from Sigma-Aldrich, St Louis, Mo.) for the endotoxin group(n=6), was equally divided and injected into the uterine wall using a 27gauge needle between the fetuses taking care not to enter the amnioticsac. This ensured that all the kits were exposed to the same amount ofendotoxin. 0.5% NaHCO₃ was infused at end of surgery and additional doseNaHCO₃ was given at 2 hour after surgery according blood gas.

Normothermia was maintained using a water circulating blanket, and heartrate, oxygen saturations, and arterial blood pressure measured through a20 G arterial catheter placed in the marginal ear artery, were monitoredcontinuously during the procedure. Maternal serum was collected beforelaparotomy (0 hours) and at 6, 24 hours following endotoxin injection.The dams were monitored daily for changes in activity, feeding andfever. A surveillance camera was placed in the rabbit room and the damsmonitored remotely to determine the time of delivery. The kits were allborn spontaneously at 31 or 32 days gestational age and the litter sizeranged from 7-12 kits. The number of live and dead kits, and weight ofall live kits was recorded.

The following animal groups were enrolled and used for this example:

Group 1: Pups exposed to 20 μg/kg E.coli LPS in utero treated withsaline I.V. (200 ul), Observe for 5 days (N=5-7)

Group 2: Pups exposed to 20 μg/kg E.coli LPS in utero treated with NACI.V. (200 ul), single dose 10 mg/kg. Observe for 5 days (N=5-7)

Group 3: Pups exposed to 20 μg/kg E.coli LPS in utero treated with NACI.V. (200 ul), single dose 100 mg/kg. Observe for 5 days (N=5-7)

Group 4: Pups exposed to 20 μg/kg E.coli LPS in utero treated withG4-PAMAM-S—S-NAC I.V (200 ul), single dose 1 mg/kg (based on preliminarydata). Observe for 5 days (N=5-7)

The dose of the saline, NAC, and G4-PAMAM-S—S-NAC used for this exampleare as follows:

a) Saline I.V (200 ul)

b) NAC I.V (200 ul), single dose 10 mg/kg.

c) NAC I.V (200 ul), single dose 100 mg/kg

d) Dendrimer-NAC I.V (200 ul), single dose 1 mg/kg

e) Dendrimer-NAC I.V (200 ul), single dose 10 mg/kg

f) Dendrimer-linker I.V (200 ul), single dose 10 mg/kg

Behavioral testing of kits administered with G4-PAMAM-S—S-NAC

All live postnatal day 1 (PND1) control and endotoxin kits from fourconsecutive litters in each group were tested to reduce the risk ofselection bias. The rabbit kits were assessed and scored for behavioraltesting, as described by Derrick et al. Briefly, the kits werevideotaped and scored on a scale of 0 (worst) to 3 (best) by two blindedobservers for (1) posture (ability to maintain prone posture), (2)righting reflex (ability to right itself from supine to prone positionfor 10 attempts), (3) activity and locomotion on a flat surface(assessed by grading the quality, intensity, and duration of spontaneousmovement of the head and front and back legs), (4) ability to move in astraight line and in circles, (5) coordination of suck and swallowassessed by feeding the rabbit kits artificially with formula from asyringe with a dropper, and (6) ability to move head during feeding(scored from 0-3 in which 0 is no movement of head and 3 is forcefulmovement of head and body). The tone on passive flexion and extensionwas assessed using the scoring based on the Ashworth scale, as describedby Derrick et al, in which 0 indicated no increase in tone and 4indicated the limb was rigid in flexion or extension.

Example 5

6 Arm-PEG-S—S-NAC Conjugate

Step 1. S-(2-thiopyridyl) N-Acetyl Cysteine

S-(2-thiopyridyl) N-Acetyl Cysteine was prepared from the reaction of 2,2¹-dithiodipyridine (5.398 g, 0.0245 mol) and NAC (2 g, 0.0122 mol) in amixture of methanol and water (1:1) stirred for 15 hours at roomtemperature. Upon completion of the reaction (monitored by TLC), most ofmethanol was removed in vacuo and the residue was dissolved in waterextracted into dichloromethane concentrated on rotavapor under reducedpressure to get crude product. Crude product was purified on silicagelcolumn chromatography with dichloromethane/methanol (8:2) gave the pureproduct as a light yellow solid (2.66 g, 0.098 mole, in 80%). Calculatedmass: ESI m/z (M+H) 273, ¹H-NMR (400 MHz, CD₃OD) δ, 1.99 (s, 3H),3.10-3.20 (m, 1H), 2.30-2.38 (m, 1H), 4.65-4.70 (m, 1H) 7.20-7.27 (m,1H, Ar), 7.80-7.85 (m, 2H Ar), 8.40-8.45 (m, 1H). ¹³C-NMR (100 MHz,CD₃OD), 21.22, 39.91, 52.05, 120.26, 121.37, 122.08, 137.98, 149.00,159.56, 172.14.

Step 2. Preparation of 6 Arm-PEG-S—S-NAC Conjugate

For the preparation of 6Arm-PEG-S—S-NAC, NAC-TP (0.245 g, 0.897 mmole)in ethanol (10 mL) was added to a solution of 6-Arm-PEG-SH (1 g, 0.1mmole) in a PBS buffered pH 7.4 (20 mL) and reaction was stirred at roomtemperature for 4 hours. The reaction was monitored with HPLC. Aftercompletion of reaction, the reaction mixture was purified using asephadex LH-20 column (Amersham Pharmacia Biotech, 3.8×45 cm) with wateras mobile phase. Water was removed under lyophilization to get purecompound in good yields (0.102 g, 0.0094 mmole, 95%). ¹H-NMR (400 MHz,CD₃OD) δ, 2.00 (s, 3H), 2.95-3.10 (m, 1H), 3.30-2.38 (m, 1H), 3.58-3.80(br, m, 4H, —OCH₂—CH₂O—) 4.40-4.50 (m, 1H), 6.95 (br, s 1H, NH amide).

Example 6

8 Arm-PEG-S—S-NAC Conjugate

Step 1. S-(2-thiopyridyl) N-Acetyl Cysteine

S-(2-thiopyridyl) N-Acetyl Cysteine was prepared from the reaction of 2,2¹-dithiodipyridine (5.398 g, 0.0245 mol) and NAC (2 g, 0.0122 mol) in amixture of methanol and water (1:1) stirred for 15 hours at roomtemperature. Upon completion of the reaction (monitored by TLC), most ofmethanol was removed in vacuo and the residue was dissolved in waterextracted into dichloromethane concentrated on rotavapor under reducedpressure to get crude product. Crude product was purified on silicagelcolumn chromatography with dichloromethane/methanol (8:2) gave the pureproduct as a light yellow solid (2.66 g, 0.098 mole, in 80%). Calculatedmass: ESI m/z (M+H) 273, ¹H-NMR (400 MHz, CD₃OD) δ, 1.99 (s, 3H),3.10-3.20 (m, 1H), 2.30-2.38 (m, 1H), 4.65-4.70 (m, 1H) 7.20-7.27 (m,1H, Ar), 7.80-7.85 (m, 2H Ar), 8.40-8.45 (m, 1H). ¹³C-NMR (100 MHz,CD₃OD), 21.22, 39.91, 52.05, 120.26, 121.37, 122.08, 137.98, 149.00,159.56, 172.14.

Step 2. Preparation of 8 Arm-PEG-S—S-NAC Conjugate

To a stirred solution of NAC-TP (0.163 g, 0.599 mmole) in ethanol (2 mL)was added a solution of 8Arm-PEG-SH (1 g, 0.05 mmol) in PBS buffered pH7.4 (20 mL) and reaction was stirred at room temperature for 4 hours.The reaction was monitored with HPLC. After completion of reaction, thereaction mixture was purified using a sephadex LH-20 column (AmershamPharmacia Biotech, 3.8×45 cm) with water as mobile phase. Water wasremoved under lyophilization to get pure compound in good yields (92%).¹H-NMR (400 MHz, CD₃OD) δ, 2.00 (s, 3H), 2.95-3.10 (m, 1H), 3.30-2.38(m, 1H), 3.58-3.80 (br m 4H, —OCH₂—CH₂O—) 4.40-4.50 (m, 1H), 6.95 (br, s1H, NH amide).

Example 7

In Vitro NAC Release Studies from PEG Conjugates

The in vitro release of NAC from the 6-Arm-PEG-S—S-NAC and8-Arm-PEG-S—S-NAC conjugates was performed in PBS (pH=7.4) at 37° C.Appropriate amounts of PEG-S—S-NAC conjugate were dissolved in releasemedia (PBS buffered) to form a solution of 1 mg/ml into eppendorf tubeand GSH was added to the conjugates to form 10 mM, 2 mM, or 2 μMconcentrations and to initiate the release of NAC. All samples were runas triplicates for statistical analysis. As control samples, conjugateswere analyzed in PBS buffered media in the absence of reducing agents.The solutions were kept at 37° C. and stirred continuously. Atpredetermined time intervals, 30 μL of samples were withdrawn andimmediately analyzed release of NAC and GS-NAC with RP-HPLC and theconcentrations of analytes were determined by using appropriatecalibrations prepared under same conditions.

Example 8

Confirmation of Antioxidative Properties of 6Arm and 8Arm PEG-S—S-NACConjugates by Reactive Oxygen Species (ROS) and Free Radical NO, andInhibition of TNF-α Production

(a) Measurement of ROS

H₂O₂ released from BV-2 cells was measured using10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), following themanufacturer's instructions. Briefly, the procedure for cell culture anddrug treatment was the same as described in previous section. Thesupernatant was mixed with 0.05 U/mL of horseradish peroxidase and 1 μMof Amplex Red in 96-well plates. After 30 min incubation, thefluorescence intensity was measured using spectrofluorometry. Excitationand emission wavelengths were 530 nm and 590 nm.

(b) NO Release Assay

Production of NO was assayed by measuring the levels of nitrite, thestable NO metabolite, in supernatant. Accumulation of nitrite insupernatant was determined by colorimetric assay with Griess reagentsystem, which uses sulfanilamide and N-(1-Naphthyl)-ethylene diamine.100 μL of the supernatant was incubated with 50 μL of Griess reagent 1(sulfanilamide) and 50 μL of Griess reagent 2N-(1-Naphthyl)-ethylenediamine for 10 min at room temperature. Theabsorbance at 540 nm was then measured, and nitrite concentration wasdetermined using a curve calibrated with nitrite standards.

(c) Detection of TNF-α

The procedure for cell culture and drug treatment was the same asdescribed in an earlier example. TNF-α secretion was measured using anELISA Kit according to the manufacturer's instruction. In brief, 50 μLof supernatant from each sample was added in 96-well ELISA plates.Biotinylated antibody reagent was applied to each well and incubated theplate at room temperature for 2 hours. After washing the plate withPBS-Tween 20, diluted streptavidin-HRP was added, and the plate wasincubated at room temperature for 30 min. After washing the plate,premixed TMB substrate solution was added. The plate was developed inthe dark for 30 min, and read at 450 nm using a microplate reader. Theconcentration of TNF-α was calculated using murine rTNF-α as standard.

Both 6-Arm-PEG-S—S-NAC conjugate and 8-Arm-PEG-S—S-NAC conjugateinhibited TNF-α production in a dose-dependent manner similar to thatobserved for free NAC at equimolar concentrations of NAC for conjugatesand free NAC, as shown in the Figures. PEG-S—S conjugates showedsignificant inhibition of nitrite production at the equivalent dose ofNAC (0.5 mM and 5 mM) when compared to the same concentration of freeNAC (as shown in the Figures) PEG-S-SNAC conjugates showed significantinhibition of ROS production at the equivalent dose of NAC (0.5 mM and 5mM) compared to the same concentration of free NAC (p<0.05 and p<0.01,respectively, as shown in the Figures.

Example 9

G4-PAMAM-O-GABA-Ethyl-S—S-Ethyl-Ampicillin

Step 1: Synthesis of Ampicillin-PDP

For the preparation of Ampicillin-PDP, SPDP (1 equivalent) in ethanol(10 mL) was added to a solution of Ampicillin (1 equivalent) in PBSbuffer pH 7.4 (20 mL) and the reaction was stirred at room temperaturefor 2 hours. The reaction was monitored with HPLC and purified on HPLCto get pure product in good yield (71%).

Step 2: Synthesis of Ampicillin-NH-Ethyl-SH

To a stirred solution of Ampicillin-PDP (1 equivalent) in PBS (pH=7.4)was added a solution of TCEP (1.5 equivalent) in PBS (pH=7.4) and thereaction was continued for 1 hour at room temperature. After completionof reaction, crude product was purified on RP-HPLC to get pure compoundin good yield.

Step 3: Synthesis of G4-PAMAM-O-GABA-Ethyl-S—S-Ethyl-Ampicillin

To a stirred solution of compound from example 3 (PAMAM-O-GABA-NH2) (1equivalent) in PBS (pH=7.4) was added ethanolic solution of SPDP (0.5equivalent) in PBS (pH=7.4) and the reaction was continued for 1 hour atroom temperature. After completion of the reaction, was addedAmpicillin-NH-Ethyl-SH and continued the reaction for 2 hours at roomtemperature. After completion of the reaction the reaction was monitoredwith HPLC. The reaction mixture was dialyzed against PBS (pH=7.4) toremove by-products and the excess of reactants and then replaced withdeionized water (3 times) dialyzed for 12 hours to remove salts. Thewater was lyophilized to get pure product in good yield.

Example 10

G4-PAMAM-O-GABA-Ethyl-S—S-Ethyl-Doxycycline

Step 1: Synthesis of Doxycycline-PDP

For the preparation of Doxycycline-PDP, SPDP (1.3 equivalent) in ethanol(10 mL) was added to a solution of Doxycycline (1 equivalent) in PBSbuffer pH 7.4 (20 mL) and the reaction was stirred at room temperaturefor 2 hours. The reaction was monitored with HPLC and purified on HPLCto get pure product in good yield.

Step 2: Synthesis of Doxycycline-NH-Ethyl-SH

To a stirred solution of Doxycycline-PDP (1 equivalent) in PBS (pH=7.4)was added a solution of TCEP (0.1.2 equivalent) in PBS (pH=7.4) and thereaction was continued for 1 hour at room temperature. After completionof reaction, crude product was purified on RP-HPLC to get pure compoundin good yield.

Step 3: Synthesis of G4-PAMAM-O-GABA-Ethyl-S—S-Ethyl-Doxycycline.

To a stirred solution of compound from example 3 (PAMAM-O-GABA-NH₂) (1equivalent) in PBS (pH=7.4) was added ethanolic solution of SPDP (1equivalent) in PBS (pH=7.4) and the reaction was continued for 1 hour atroom temperature. After completion of the reaction, was addedDoxycycline-NH-Ethyl-SH and continued the reaction for 2 hours at roomtemperature. After completion of the reaction the reaction was monitoredwith HPLC. The reaction mixture was dialyzed against PBS (pH=7.4) toremove by-products and the excess of reactants and then replaced withdeionized water (3 times) dialyzed for 12 hours to remove salts. Thewater was lyophilized to get pure product in good yield.

Example 11

PAMAM-NH-Ethyl-S—S-Ethyl-CO—NH-GABA-O-Dexamethasone

Step 1: Synthesis of Dexamethasone-O-GABA-BOC

A solution of BOC-GABA-OH (mg, mmol)) in DMF (3:1) was cooled to 0° C.and then treated with a solution of EDC (mg, mmol), DMAP (mg, mmol) andDexamethasone, The reaction was stirred at room temperature for 24hours. The reaction mixture was purified on silicagel columnchromatography with ethyl acetate hexane as eluent to get pure compound.

Step 2: Synthesis of Dexamethasone-O-GABA-NH₂

To a stirred solution of Dexamethasone-O-GABA-BOC (1 g) was treated withtrifloroacetic acid and dichloromethane (1:1, 10 mL). The reaction wasstirred at room temperature for 10 min. After completion of the reactiontrifloroacetic acid/dichloromethane was removed under rotavapor.Reaction mixture was neutralized with PBS (pH=7.4) and purified onsilicagel column chromatography with ethyl acetate hexane as eluent toget pure compound.

Step 3: Dexamethasone-O-GABA-NH—CO-PDP

SPDP (1.2 equivalent) in ethanol (10 mL) was added to a solution ofDexamethasone-O-GABA-NH₂ (1 equivalent) in PBS buffer pH 7.4 (20 mL) andthe reaction was stirred at room temperature for 2 hours. Aftercompletion of reaction compound was extracted into ethyl acetate,solvent was evaporated under reduced pressure to get crude product. Thecrude product was purified on silicagel column chromatography with ethylacetate and hexane as eluent to get pure compound in good yield.

Step 4 PAMAM-O-GABA-NH—CO-PDP

For the preparation of PAMAM-O-GABAB-NH—CO-PDP, to a stirred solution ofcompound from example 3 (PAMAM-O-GABA-NH₂) (1 equivalent) in PBS(pH=7.4) SPDP (1.2 equivalent) in ethanol (10 mL) was added to asolution of PBS buffer pH 7.4 (20 mL) and PAMAM-O-GABA-NH₂ dendrimer (1equivalent) to provide sufficient modification whilst preventing loss ofproduct due to the precipitation of highly modified dendrimer. Reactionwas stirred at room temperature for 2 hours. The reaction was monitoredwith HPLC. After completion of reaction, the reaction mixture wasdialyzed against DMSO remove by-products and the excess of reactants andthen lyophilized to get pure product in good yield (71%).

Step 5: PAMAM-O-GABA-NH—CO-Ethyl-SH

To a stirred solution of PAMAM-NH—CO-PDP (1 equivalent) in PBS (pH=7.4)was added a solution of TCEP (1.2 equivalent) in PBS (pH=7.4) and thereaction was continued for 1 hour at room temperature. After completionof reaction, crude product was purified on RP-HPLC to get pure compoundin good yield.

Step 6: Synthesis ofPAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-CO—NH-GABA-O-Dexamethasone

To a stirred solution of Dexamethasone-O-GABA-NH-PDP (1 equivalent) inPBS (pH=7.4) was added PAMAM-NH-Ethyl-SH (1 equivalent) and the reactionwas continued for 2 hours at room temperature. After completion of thereaction the reaction was monitored with HPLC. The reaction mixture wasdialyzed against PBS (pH=7.4) to remove by-products and the excess ofreactants and then replaced with deionized water (3 times) dialyzed for12 hours to remove salts. The water was lyophilized to get pure productin good yield.

Example 12

PAMAM-O-GABA-NH-Ethyl-S—S-Ethyl-CO—NH-GABA-O-Indomethacin Carrier (VII)

Step 1: Synthesis of Indomethacin-O-GABA-NH₂

To a stirred solution of Indomethacin-O-GABA-BOC (1 g) was treated withtrifloroacetic acid and dichloromethane (1:1, 10 mL). The reaction wasstirred at room temperature for 1 hour. After completion of the reactiontrifloroacetic acid/dichloromethane was removed under rotavapor.Reaction mixture was neutralized with PBS (pH=7.4) and purified onsilicagel column chromatography with ethyl acetate hexane as eluent toget pure compound.

Step 2: Indomethacin-O-GABA-NH—CO-PDP

Solution of SPDP (1.2 equivalent) in ethanol (10 mL) was added to asolution of Indomethacin-O-GABA-NH₂ (1 equivalent) in PBS buffer pH 7.4(20 mL) and the reaction was stirred at room temperature for 2 hours.After completion of reaction compound was extracted into ethyl acetate,solvent was evaporated under reduced pressure to get crude product. Thecrude product was purified on silicagel column chromatography with ethylacetate and hexane as eluent to get pure compound in good yield.

Step 3: PAMAM-O-GABA-NH-Ethyl-S—S-GABA-Indomethacin

To a stirred solution of compound from example 11(PAMAM-O-GABAB-NH—CO-Ethyl-SH) (1 equivalent) in PBS (pH=7.4) was addedto a solution of Indomethacin-O-GABA-NH-PDP (1 equivalent) in PBS bufferpH 7.4 (20 mL) and continued the reaction for 2 hours at roomtemperature. After completion of the reaction, the reaction wasmonitored with HPLC. The reaction mixture was dialyzed against PBS(pH=7.4) to remove by-products and the excess of reactants and thenreplaced with deionized water (3 times) dialyzed for 12 hours to removesalts. The water was lyophilized to get pure product in good yield.

Example 13

PAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH—NH-Progesterone Carrier (VII)

Step 1: Synthesis of Progesterone-PDPH

Solution of PDPH (2 equivalent) in DMSO (10 mL) was added to a solutionof Progesterone (1 equivalent) and the reaction was stirred at roomtemperature for 12 hours. After completion of reaction compound wasextracted into ethyl acetate, solvent was evaporated under reducedpressure to get crude product. The crude product was purified onsilicagel column chromatography with ethyl acetate and hexane as eluentto get pure compound in good yield.

Step 2: PAMAM-O-GABA-NH—CO-Ethyl-S—S-GABA-Progesterone

To a stirred solution of compound from example 12(PAMAM-O-GABA-NH—CO-Ethyl-SH) (1 equivalent) in PBS (pH=7.4) was addedto a solution of Progesterone-PDPH (1 equivalent) n PBS buffer pH 7.4(20 mL) and continued the reaction for 2 hours at room temperature.After completion of the reaction, the reaction was monitored with HPLC.The reaction mixture was dialyzed against PBS (pH=7.4) to removeby-products and the excess of reactants and then replaced with deionizedwater (3 times) dialyzed for 12 hours to remove salts. The water waslyophilized to get pure product in good yield.

Example 14

PAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹(SEQ ID NO: 1)

Step 1: Synthesis of Boc-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹(SEQ ID NO:1)

A solution of BOC-GABA-OH (1.5 equivalent)) in DMF (3:1) was cooled to0° C. and then treated with a solution of EDC (1.5 equivalent), DMAP(0.01 equivalent) and 5¹-AGUCGGAGGCUUAAUUACA-3′ (SEQ ID NO: 1) and thereaction was stirred at room temperature for 24 hours. The reactionmixture was purified on HPLC to get pure compound.

Step 2: Synthesis of NH₂-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹ (SEQ ID NO: 1)

To a stirred solution of Boc-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹ (SEQ IDNO: 1) (1 equivalent) was treated with trifloroacetic acid anddichloromethane (1:1, 20 equivalent). The reaction was stirred at roomtemperature for 1 hour. After completion of the reaction trifloroaceticacid/dichloromethane was removed under rotavapor. Reaction mixture wasneutralized with PBS (pH=7.4) and the reaction mixture was purified onHPLC to get pure compound.

Step 3: PDP-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹ (SEQ ID NO: 1)

Solution of SPDP (1.2 equivalent) in ethanol (10 mL) was added to asolution of NH₂-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹ (SEQ ID NO:1) (1equivalent) in PBS buffer pH 7.4 (2 mL) and the reaction was stirred atroom temperature for 2 hours. After completion of the reaction thereaction mixture was purified on HPLC to get pure compound.

Step 4:AMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹(SEQ ID NO: 1)

To a stirred solution of compound from example 12(PAMAM-O-GABAB-NH—CO-Ethyl-SH) (1 equivalent) in PBS (pH=7.4) was addedto a solution of PDP-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹(SEQ ID NO: 1)(1 equivalent) in PBS buffer pH 7.4 (20 mL) and continued the reactionfor 2 hours at room temperature. After completion of the reaction, thereaction mixture was purified on HPLC to get pure compound.

Example 15

PAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹(SEQ ID NO: 2)

Step 1: Synthesis of Boc-NH-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹ (SEQ ID NO:2)

A solution of BOC-GABA-OH (1.5 equivalent)) in DMF (3:1) was cooled to0° C. and then treated with a solution of EDC (1.5 equivalent), DMAP(0.01 equivalent) and 5¹-CAGGAAAUUUGCCUAUUGA-3′ (SEQ ID NO: 2) and thereaction was stirred at room temperature for 24 h. The reaction mixturewas purified on HPLC to get pure compound.

Step 2: Synthesis of NH₂-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹ (SEQ ID NO:2)

To a stirred solution of Boc-NH-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹ (SEQ IDNO: 2) (1 equivalent) was treated with trifloroacetic acid anddichloromethane (1:1, 20 equivalent). The reaction was stirred at roomtemperature for 1 hour. After completion of the reaction trifloroaceticacid/dichloromethane was removed under rotavapor. Reaction mixture wasneutralized with PBS (pH=7.4) and the reaction mixture was purified onHPLC to get pure compound.

Step 3: PDP-NH-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹ (SEQ ID NO: 2)

Solution of SPDP (1.2 equivalent) in ethanol (10 mL) was added to asolution of NH₂-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹ (SEQ ID NO: 2) (1equivalent) in PBS buffer pH 7.4 (2 mL) and the reaction was stirred atroom temperature for 2 hours. After completion of the reaction thereaction mixture was purified on HPLC to get pure compound.

Step 4:AMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-CAGGAAAUUUGCCUAUUGA-3¹(SEQ ID NO: 2)

To a stirred solution of compound from example 12(PAMAM-O-GABAB-NH—CO-Ethyl-SH) (1 equivalent) in PBS (pH=7.4) was addedto a solution of PDP-NH-GABA-O-5¹-AGUCGGAGGCUUAAUUACA-3¹ (SEQ ID NO: 1)(1 equivalent) in PBS buffer pH 7.4 (20 mL) and continued the reactionfor 2 hours at room temperature. After completion of the reaction, thereaction mixture was purified on HPLC to get pure compound.

Example 16

PAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹(SEQ ID NO: 3)

Step 1: Synthesis of Boc-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO:3)

A solution of BOC-GABA-OH (1.5 equivalent)) in DMF (3:1) was cooled to0° C. and then treated with a solution of EDC (1.5 equivalent), DMAP(0.01 equivalent) and 5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3) and thereaction was stirred at room temperature for 24 hours. The reactionmixture was purified on HPLC to get pure compound.

Step 2: Synthesis of NH₂-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3)

To a stirred solution of Boc-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ IDNO: 3) (1 equivalent) was treated with trifloroacetic acid anddichloromethane (1:1, 20 equivalent). The reaction was stirred at roomtemperature for 1 hour. After completion of the reaction trifloroaceticacid/dichloromethane was removed under rotavapor. Reaction mixture wasneutralized with PBS (pH=7.4) and the reaction mixture was purified onHPLC to get pure compound.

Step 3: PDP-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3)

Solution of SPDP (1.2 equivalent) in ethanol (10 mL) was added to asolution of NH₂-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3) (1equivalent) in PBS buffer pH 7.4 (2 mL) and the reaction was stirred atroom temperature for 2 hours. After completion of the reaction thereaction mixture was purified on HPLC to get pure compound.

Step 4:PAMAM-O-GABA-NH—CO-Ethyl-S—S-Ethyl-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹(SEQ ID NO: 3)

To a stirred solution of compound from example 12(PAMAM-O-GABAB-NH—CO-Ethyl-SH) (1 equivalent) in PBS (pH=7.4) was addedto a solution of PDP-NH-GABA-O-5¹-UAAGGACCAAGACCAUCCA-3¹ (SEQ ID NO: 3)(1 equivalent) in PBS buffer pH 7.4 (20 mL) and continued the reactionfor 2 hours at room temperature. After completion of the reaction, thereaction mixture was purified on HPLC to get pure compound.

Example 17

Permeability of G4-PAMAM-FITC Across the Rabbit Amniotic Membrane

The permeability of the G4-PAMAM-FITC across the normal rabbit amnioticmembrane and endotoxin treated rabbit amniotic membrane was studiedusing a side by side Permegear diffusion chamber at 37° C. for 48 hours.Endotoxin treated membranes were used to mimic the condition of E. coliinfection in uterus. The freshly excised rabbit membranes obtained aftersacrificing the rabbit was placed in between the donor and receptorchamber. The donor chambers were filled with 3 ml of FITC (0.9 mg/ml)and G4-PAMAM-FITC (3 mg/ml) solution in sterile PB buffer pH 7.4respectively and samples were collected from the receptor chamber filledwith 3 ml sterile PB buffer pH 7.4 at regular intervals and analyzed byUV and fluorescent plate reader. The permeation of dendrimer(G4-PAMAM-FITC) was compared against the small molecule (FITC alone).

The permeation of the G4-PAMAM-FITC was significantly lower than theFITC. 50% of FITC crossed the membrane in 1 hour as compared to theG4-PAMAM-FITC, which crossed 17% in 1 hour.

Example 18

Anti-Inflammatory and Anti-Oxidant Activity of AnionicDendrimer-N-Acetyl Cysteine Conjugates in Activated Microglial Cells

Perinatal brain damage is a major cause of disability and death ininfants. A significant fraction of babies who suffer brain damage duringand around birth develop cerebral palsy. There is increasing evidencesuggesting that infection involving the uterus during pregnancy can leadto cerebral palsy in the baby (Makki et al., 2008; Romero et al., 1998,2006, 2007a,b; Gomez et al., 2007). Recent studies demonstrate that themain mechanism of brain damage is due to the activation of microglialcells in the fetal brain that release inflammatory markers leading tothe death of normal brain cells. These activated cells are not normallyfound in the brain. Infection or inflammation can activate microglialcells and cause them to migrate to the brain where they damage thenormal brain cells. Therefore, developing intracellular drug deliverystrategies to deliver drugs to activated microglial cells may help indecreasing the neuroinflammation and in the attenuation of the whitematter injury. However, diagnosis and drug therapy during pregnancy isstill a challenge. Recent work on a pregnant rabbit model has been ableto successfully capture neuroinflammation-induced cerebral palsy, andits treatment using an anti-inflammatory drug, N-acetyl cysteine.

Developments in the rapidly expanding field of nanomedicine are offeringa variety of nanoscale delivery vehicles such as liposomes,nanoparticles, and dendrimers (Lee et al., 2005; Cheng et al., 2008;Villalonga-Barber et al., 2008). Dendrimers are monodisperse, tree-likepolymers with a large density of tailorable, functional groups that havepotential to deliver drugs in a targeted manner to the site of action(Wolinsky and Grinstaff, 2008). Their nanoscale branching architecturesize (˜5 nm) enables them to be transported into cells. When this iscombined with appropriate targeting mechanism and intracellular drugrelease profiles, conjugates of dendrimers can be potentially potent fora variety of therapeutic applications. Anionic PAMAM dendrimers arebeing explored as drug delivery vehicles in this study (Wiwattanapatapeeet al., 2004). In addition to being highly non-cytotoxic compared to thecationic dendrimers, anionic dendrimers have shown to be highlyeffective in transcellular transport and has been used for oral deliveryapplications. Previous studies in cancer cells have also shown thatefficacy of anionic PAMAM dendrimer-methotrexate (MTX) conjugates weresignificantly better than cationic PAMAM dendrimer-MTX conjugates(Gurdag et al., 2005). This difference has been at least partiallyattributed to differences in lysosomal residence times and intracellulardrug release from anionic and cationic dendrimer-drug conjugates. Therewas previously reported the synthesis, efficacy, and drug release fromcationic PAMAM-generation-4 dendrimer-N-acetyl-L-cysteine conjugates,where the conjugate showed a significantly better efficacy than the freedrug, perhaps due to superior intracellular transport of the drug by thedendrimer, and its subsequent rapid release from the glutathionesensitive disulfide linker (Navath et al., 2008). Anionic PAMAMdendrimers may be more effective in vivo platforms compared to cationicPAMAM dendrimers for drug delivery applications, because of their bettercytotoxicity profiles, and reduced protein binding (Malik et al., 2000).The efficacy of the anionic dendrimer conjugates will be compared withthose of the previous conjugates, where other drugs (e.g. methotrexateand methyl prednisolone) were investigated (Khandare et al., 2005; Kolheet al., 2003, 2006; Kannan et al., 2004).

N-acetyl-L-cysteine (NAC) is an anti-inflammatory and antioxidant agentused in a wide range of clinical applications (Wang et al., 2007). It isbeing explored for use in neuroinflammation in perinatal applications(Paintlia et al., 2008). NAC could effectively block CD11 b expressionin mouse BV-2 cells and primary microglia, which is correlated to theseverity of microglial activation in various neuroinflammatory diseasesreported (Roy et al., 2008). However, early pharmacokinetic studiessuggested that oral NAC bioavailability was low, between 6% and 10%, dueto low blood concentration of NAC. The biological half-life of NAC isonly 1.5 hours in the blood stream. Building on the recent findings thatsuggest that PAMAM dendrimers can target neuroinflammation, even afterintravenous administration, this study seek build conjugates byunderstanding the efficacy in target cells (Kannan et al., 2007).Specifically, the anti-inflammatory and anti-oxidative effects of PAMAMdendrimer-NAC conjugate, compared to free NAC, were investigate onactivated microglial cells, which are the target cells for this drug invivo. The unique aspect of this study arises from the fact that theactivity of the conjugated drug is being explored using multiple assays,for dendrimer-drug conjugates in non-cancer applications.

Synthesis of PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

PAMAM-(COOH)₄₆-(NAC)₁₈ was prepared in three steps. n the first step,S-(2-thiopyridyl) glutathione was prepared from the reaction of2-2¹-dithiodipyridine in excess and the corresponding peptide in amixture of methanol and water at room temperature. Upon completion ofthe reaction, methanol was removed in vacuo and the residue was washedwith dichloromethane. The aqueous solution was subjected to reversephase (RP)-HPLC purification, and lyophilization of the eluent gave thepure product as a white solid. In the second step, the above compoundwas reacted with NAC (1 eq) in PBS in pH 7.4 to get desired GS-NACintermediate and purified. In third step, to introduce the GS-NAC,PAMAM-COOH was reacted with GS-NAC (64 eq/dendrimer) in the presence ofPyBop/DIEA to give desired PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate.Introduction of 18 GS-NAC was confirmed using HPLC, ¹H NMR (FIG. 51) andMALDI. The MALDI analysis of the PAMAM-(COOH)₄₆-(NAC)₁₈ conjugatesuggested a molecular weight of 19.7 kDa (18 GS-S-NAC molecules on onePAMAM-COOH dendrimer, Table 2). The attachment of GS-S-NAC groups to thedendrimer was also confirmed using ¹H NMR analysis, as evidenced by theappearance of methyl protons at 1.70, 1.92 ppm that indicate theformation of GS-S-NAC conjugate with dendrimer.

Cell Culture

Mouse microglial cell line (BV-2) was obtained from Children's Hospitalof Michigan Cell Culture Facility. Cells were grown in 75 mm² cultureflasks using Dulbecco's Modified Eagle Medium (DMEM) supplemented with5% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37° C.with 5% CO₂ in an incubator. The cells were subcultured every 48 hoursand harvested from subconfluent cultures (60-70%) using 0.05%trypsin-EDTA.

Cells Treatment with PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

BV-2 cells (passage 16) were seeded in 24 well plates at 10⁵/mL/well andincubated for 24 hours. The medium was removed, and the cells wereexposed to 100 ng/mL of LPS and varying concentrations ofPAMAM-(COOH)₄₆-(NAC)₁₈ conjugate in 500 μL of serum free medium for 3hours. The medium was removed again, and 500 μL of fresh serum freemedium containing 100 ng/mL of LPS was added and incubated for 24 hoursand 72 hours. Control treatment with varying concentrations of free NAC,positive control with 100 ng/mL of LPS induction, but negative controlwithout any LPS induction and treatment were also studied. The culturemedium was collected at specific time intervals of 24 hours and 72hours, and spun at 1500 rpm for 5 min. The supernatant was stored at−80° C. for further assays.

Measurement of ROS

H₂O₂ released from BV-2 cells was measured using10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), following themanufacturer's instructions (Alexandre et al., 2006; Min et al., 2003).The procedure for cell culture and drug treatment was the same asdescribed in previous section. The supernatant was mixed with 0.05 U/mLof horseradish peroxidase and 1 μM of Amplex Red in 96-well plates.After 30 min incubation, the fluorescence intensity was measured usingspectrofluorometry. Excitation and emission wavelengths were 530 nm and590 nm respectively.

NO Release Assay

Production of NO was assayed by measuring the levels of nitrite, thestable NO metabolite, in the culture medium. Accumulation of nitrite inthe medium was determined by colorimetric assay with Griess reagentsystem, which uses sulfanilamide and N-(1-Naphthyl)-ethylene diamine.From the treated cells in the medium, 100 μL of the supernatant wasincubated with 50 μL of Griess reagent 1 (sulfanilamide) and 50 μL ofGriess reagent 2 N-(1-Naphthyl)-ethylenediamine for 10 min at roomtemperature. The absorbance at 540 nm was then measured, and nitriteconcentration was determined using a calibration curve prepared usingnitrite standards.

Detection of TNF-α

The procedure for cell culture and drug treatment was the same asdescribed in previous section. TNF-α secretion was measured using anELISA kit according to the manufacturer's instructions. In brief, 50 μLof supernatant from each sample was added in 96-well ELISA plates.Biotinylated antibody reagent was applied to each well and the plate wasincubated at room temperature for 2 hours. After washing the plate withPBS-Tween 20, diluted streptavidin-HRP was added, and the plate wasincubated at room temperature for 30 min. After washing the plate, thepremixed TMB substrate solution was added. The plate was developed inthe dark for 30 min, and read at 450 nm using a microplate reader. Theconcentration of TNF-α was calculated using murine rTNF-α as standard.

Statistical Analysis

Data are presented as mean±SD. Specific comparisons between control andindividual experiment were analyzed by Student's t-test with P-valueless than 0.05 considered as statistical significance.

Results

Preparation and Characterization of Dendrimer-NAC Conjugates

A PAMAM dendrimer conjugate [PAMAM-(COOH)₄₆-(NAC)₁₈] has been developed,using a disulfide linker, for glutathione (GSH)-mediated intracellularrelease of NAC. To facilitate the linking of NAC to dendrimer viadisulfide bond spacer group, glutathione (GSH) were used. To prepareGS-NAC, GSH was reacted with 2,2¹-dithiodipyridine to give GS-TP, whichwas further reacted with NAC to give GS-NAC. To introduce the GS-NAC,PAMAM-COOH was reacted with GS-NAC in the presence of PyBop/DIEA to givethe desired PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate.

PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

PAMAM-(COOH)₄₆-(NAC)₁₈ conjugates having cleavable disulfide linkagesare designed for intracellular delivery based on glutathione levels.PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate was synthesized using a three-stepsequence. S-(2-thiopyridyl) glutathione was prepared from the reactionof 2,2¹-dithiodipyridine and GSH, and purified through HPLC. Thiscompound was reacted with NAC in PBS (pH 7.4) to get the desiredGlutathione-N-Acetyl Cysteine (GS-S-NAC) intermediate upon purification.The formation of disulfide bond was confirmed by ¹H NMR and ESI-MS.Appearance of methyl groups in ¹H NMR at 1.90 ppm indicates theformation of disulfide bond between the GSH and NAC. To introduce theGS-SNAC, PAMAM-COOH was reacted with GS-S-NAC in the presence ofPyBop/DIEA to obtain the desired PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate (FIG.51). Introduction of GS-S-NAC was confirmed HPLC, ¹H NMR and MALDI.MALDI analysis yielded a molecular weight of 19.7 kDa (FIG. 51) (18GS-S-NAC molecules for one molecule of PAMAM-COOH dendrimer). The numberof GS-S-NAC groups was also determined using H NMR analysis (FIG. 51),with the appearance of methyl protons at 1.70, 1.92 ppm indicating theformation of GS-NACconjugate with dendrimer. The ¹H NMR and the MALDIdata for the drug payload agree very well with each other, as summarizedin Table 1.

TABLE 1 Molecular weight estimation (by MALDI-TOF, and ESI-MS) of NAC,FITC in PAMAM-(COOH)₄₆-(NAC)₁₈, (COOH)₆₂-(FITC)₂, respectively. Name ofthe Molecular Pay Purity of Solubility in compound weight load conjugatePBS/H₂O GS-S-NAC  468 kDa — 99.1% Soluble PAMAM-- 19.7 kDa 18 99.5%Highly (COOH)₄₆-(NAC)₁₈ soluble FITC  389 kDa — 99.5% Not soluble PAMAM-13.7 kDa 37 99.5% Highly CO—NH—CH₂—NH₂ soluble PAMAM- ~14.7 kDa   299.5% Highly (COOH)₆₂- soluble (FITC)₂Release of NAC from PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

N-acetyl cysteine release from PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate wasanalyzed at intracellular GSH concentration (10 mM). The detailedmechanism and kinetics of the drug release have been described elsewhere(Kurtoglu et al., 2009). Briefly, the results suggest that the conjugatewas able to release significant amounts of free NAC within an hour, inthe presence of GSH. In the absence of GSH, or at GSH levels in theblood (20 μM), no drug release was seen. PAMAM-(COOH)₄₆-(NAC)₁₈conjugate released 39% of NAC in the free form and another 6% in theGS-S-NAC form within 1 hours, yielding a total of 45% NAC release. Theeventual application, where neuroinflammation in the newborn rabbit pupsis treated with the conjugates, requires relatively fast release of NACfrom the conjugates. The release is desired over a period of a few days.The timescales for the cellular efficacy has been chosen to be 24 hoursor 72 hours, with this in vivo requirement in mind.

Anti-Oxidative Activity of PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

The anti-oxidative properties of the conjugate were tested by measuringthe reactive oxygen species (ROS) and free radical NO in activatedmicroglial cells. This is an indication of the ability of the conjugatesto treat neuroinflammation, since these cells play a central role in thedisease process. In prior studies in activated cells, it has beenobserved that ROS and NO production at 72 hours after activation wassignificantly higher than that at 24 hr after activation. This is alsoseen in the studies (FIGS. 52 and 53).

ROS Assay

ROS has been known to play important roles in oxidation andinflammation. PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate inhibited the release ofROS induced by LPS in BV-2 cells. After 24 hours of stimulation with LPSfollowing 3 hours pre-treatment, free NAC did not affect ROS productionover a concentration range of 0.5-8 mM (P>0.05). In contrast, thePAMAM-(COOH)₄₆-(NAC)₁₈ conjugate showed significant inhibition of ROSproduction at 2 mM and 8 mM when compared to the same concentration offree NAC. After 72 hours of activation with LPS following 3 hourspre-treatment, only the highest concentration of free NAC (8 mM)inhibited ROS release moderately (30%), whereas the lowest concentrationof PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate (0.5 mM) showed inhibition of ROSproduction (25%). The conjugate significantly inhibited ROS productionat 8 mM when compared to the same concentration of free NAC (68%). Theinhibition showed a dose-dependent response. The correspondingconcentrations of PAMAM-COOH dendrimer did not affect the cells ROSproduction after 24 hours and 72 hours stimulation of LPS following 3hours pre-treatment (FIG. 52, Table 2).

Nitrite Assay

After 24 hours of activation with LPS following 3 hours pre-treatment,only the highest concentration of free NAC (8 mM) significantly reducednitrite release (˜70%), though there was a dose-dependent response. Incontrast, the PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate reduced nitrite releaseat the lowest equivalent dose of NAC (0.5 mM)(˜61%). The conjugatesignificantly reduced nitrite release at all the three equivalentconcentrations compared to free NAC. After 72 hours of activation withLPS following 3 hours pre-treatment, free NAC reduced nitrite release ina dose-dependent manner. PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate showedsignificant reduction of nitrite release even at the lowestconcentration (0.5 mM) when compared to the same concentration of freeNAC (by ˜60%). In fact, 0.5 mM NAC in the conjugated form, showed betterefficacy compared to 2 mM of free NAC. The conjugate showed adose-dependent response (FIG. 53, Table 2). The free PAMAM-COOHdendrimer control slightly decreased the nitrite release only at thehighest concentration (0.44 mM) after 72 hours stimulation of LPSfollowing 3 hours pre-treatment (FIG. 54, Table 2).

Anti-Inflammatory Activity of PAMAM-(COOH)₄₆-(NAC)₁₈ Conjugate

Anti-inflammatory activity of PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate wasevaluated in vitro using BV-2 cells, which were activated with LPS toinduce TNF-α synthesis. In contrast to ROS and NO levels afteractivation, TNF-α levels have been shown to be appreciably faster, withsignificant increases at 24 hours (Waseem et al., 2008; El-Remessy etal., 2008). This is consistent with the present study, where high TNF-αlevels were seen after 24 hours. After 24 hours and 72 hours ofactivation with LPS following 3 hours pre-treatment, free NAC inhibitedTNF-α production in a dose-dependent manner, with a maximum reduction of˜45% at 8 mM concentration. In comparison, the PAMAM-(COOH)₄₆-(NAC)₁₈conjugate reduced the TNF-α production very significantly (˜67%) even atthe lowest equivalent dose of NAC (0.5 mM). Typically, the conjugateshowed better efficacy at 0.5 mM compared to free NAC at 8 mM in all thethree assays. The inhibitory effect did not show a significant dosedependence (FIG. 55, Table 2), perhaps because appreciable reduction wasseen even at the lowest dose. The free PAMAM-COOH dendrimer control didnot inhibit TNF-α production (FIG. 56, Table 2).

TABLE 2 Inhibitory rate of NAC, conjugate and dendrimer in markers ofoxidative stress and inflammation after 72 hours stimulation of LPSfollowing 3 hours treatment. H₂O₂ reduction Nitrite TNF-α Drug dose (%)reduction ( %) reduction (%) NAC  0.5 mM  5.54 ± 6.38 10.52 ± 6.43 34.98± 2.43   2 mM 22.28 ± 8.33 34.66 ± 3.22 35.13 ± 4.44   8 mM 41.31 ± 2.3372.70 ± 5.56 44.57 ± 4.35 Dendrimer 0.03 mM  2.09 ± 14.53 −20.16 ± 12.90−10.21 ± 5.07  0.11 mm  −9.37 ± 24.63  38.29 ± 17.74 −5.82 ± 4.58 0.44mM  −3.16 ± 21.02  46.77 ± 15.32  8.64 ± 6.73 D-NAC  0.5 mM 30.81 ± 8.0860.82 ± 6.05 67.46 ± 3.91   2 mM 51.13 ± 4.93 64.85 ± 5.12 74.43 ± 3.54  8 mM 68.75 ± 4.14 75.75 ± 1.85 77.37 ± 3.31Discussion

An anionic dendrimer-NAC conjugate was prepared with a high drugpayload. The drug was linked to the dendrimer using a GSH sensitivelinker, which released the drug at intracellular GSH concentrations. Thecell uptake and the anti-oxidant and anti-inflammatory activity wereevaluated in activated microglial cells, which are the target cells forthe in vivo application in a rabbit model of cerebral palsy.

From the results of flow cytometry and confocal microscopy, it appearsthat PAMAM-(COOH)₆₂-(FITC)₂ dendrimer are transported inside the cellsefficiently and relatively rapidly. BV-2 cells are known to possessanionic charge, which is the same as that of PAMAM-(COOH)₆₂-(FITC)₂dendrimer at physiological pH. Therefore, it may be expected that thecellular entry of PAMAM-(COOH)₆₂-(FITC)₂ into BV-2 cells may berestricted. Despite this, the cell uptake is significant, perhapssuggesting an active endocytosis mechanism (Kannan et al., 2007; Perumalet al., 2008).

Cytotoxicity assay demonstrated that free NAC, free dendrimer, and thePAMAM-(COOH)₄₆-(NAC)₁₈ conjugate are relatively nontoxic. Previous workon the cytotoxicty of dendrimers has suggested that the toxicity dependson end functionality, concentration and the time of exposure (Malik etal., 2000). Typically, anionic dendrimers have found to be significantlyless toxic than cationic dendrimers. For example, Malik et al. (2000)observed that the PAMAM-G2.5-COOH dendrimer did not exhibit anysignificant toxicity against B16F10 melanoma cells at 2 mg/mL. The factthat microglial cells do not show measurable cytotoxicity at theselevels, allows researchers to assess the efficacy of the nanodevices atwell-defined treatment conditions.

Inflammatory responses in the brain are now thought to be mainlyassociated with activity of microglial cells, the resident macrophagesof CNS, serving the role of immune surveillance and host defense undernormal condition. Under pathological conditions, microglial cells becomeactivated and have been implicated as the predominant cell typegoverning inflammation-mediated neuronal damage. In particular,activated microglial cells exert cytotoxic effects by releasinginflammatory mediators, such as reactive oxygen species (ROS), nitricoxide (NO) and a variety of proinflammatory cytokines such as tumornecrosis factor alpha (TNF-α). In this study, LPS was used to activateBV-2 microglial cells in vitro. LPS, the cell wall component ofGram-negative bacteria, is known to activate nitrogen-activated proteinkinases, nuclear factor kB (NF-kB), protein kinase C and tyrosinekinases, which have been implicated in the release of immune-relatedcytotoxic factors, such as ROS, NO and proinflammatory cytokines (Lu etal., 2007).

In the in vivo studies, N-acetyl-L-cysteine (NAC) was used to addressneuroinflammation in perinatal brain injury (Makki et al., 2008).Therefore, the cellular efficacy of the conjugate in activated BV-2microglial cells was evaluated. The anti-oxidative properties of theconjugate were tested by measuring the ROS and NO levels in cell culturemedium, and nitrite was chosen as a marker of free radical NO. Theanti-inflammatory activity was evaluated by measuring the TNF-α level incell culture medium. The efficacy of PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate onanti-oxidation and anti-inflammation between 24 hours or 72 hoursstimulation of LPS following 3 hours pre-treatment were compared.

In the experiment, production of ROS by dysfunctional mitochondria or byxanthine oxidase may contribute to LPS-induced oxidative stress withmicroglial cells (Paintlia et al., 2007). Peroxisomes are important fordetoxification of ROS, and LPS induced effects are known to causeperoxisomal dysfunction that has been linked with ROS generation inapoptosis (Paintlia et al., 2007). NAC can abolish LPS-induced ROSproduction. The PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate (8 mM) showedsignificant therapeutic effect in reducing the ROS release compared tothe same concentration of free NAC after 72 hours stimulation of LPSfollowing 3 hours pre-treatment. Dendrimer did not show any effects onROS release following short and long time treatment, suggesting that theconjugate is able to transport and release the drug inside the cells.

Nitric oxide (NO) is produced by most cells, and is cytotoxic at highconcentration or in the presence of superoxide. The cytotoxic effectsare due, at least in part, to the formation of peroxynitrite from NO andsuperoxide, which represents a strong oxidant and nitrating agent. Onthat other hand, NO itself can exert cytotoxic effects due tonitrosylation reaction and the inhibition of the mitochondrialrespiration by binding to the mitochondrial cytochrome c oxidase (Noacket al., 2000). NAC can suppress LPS-induced NO production. ThePAMAM-(COOH)₄₆-(NAC)₁₈ conjugate appears to show better therapeuticeffect towards inhibiting activated microglial cells from releasing NOwhen compared to the same concentration of free NAC after 24 hours and72 hours stimulation of LPS following 3 hours pre-treatment. Highconcentration of free PAMAM-COOH dendrimer significantly decrease theconcentration of nitrite in medium. The mechanism is perhaps by thebinding of the interior secondary amines between dendrimer and nitrite.Therefore, the PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate may be decreasing thenitrite level in cell culture medium through the effects of both NAC anddendrimers.

LPS can also stimulate the secretion of pro-inflammatory cytokinesTNF-α, IL-1β and IL-6 in maternal and fetal compartments including fetalbrain. Pro-inflammatory cytokines induced severe peroxisomal dysfunctionand increased oxidative stress. Anti-inflammatory effects of NAC areattributed to the suppression of pro-inflammatory cytokine expressionand release, adhesion molecule expression and activation of NF-κB incells (Paintlia et al., 2008). The PAMAM-(COOH)₄₆-(NAC)₁₈ conjugateshowed more significant efficacy to inhibit activated microglial cellsto release TNF-α when compared to the same concentration of free NACafter 24 hours and 72 hours stimulation of LPS following 3 hourspre-treatment. PAMAM-COOH dendrimer did not reduced TNF-α release.

From these results, it appears that high drug payload in the dendrimerconjugate produces a high local drug concentration inside the cells. Theutilization of a GSH sensitive release mechanism is enabling faster drugrelease and higher pharmacological response compared to the sameconcentration of free drug. The improved in vitro efficacy of theconjugates is a significant result, since most polymer-drug conjugatesshow less efficacy in cells (partly attributed to slower, inefficientdrug release from the conjugates), even though enhanced permeation andretention effect (EPR) and ligand-targeting produces better efficacy invivo (references). Specific to dendrimers, recent studies have shownthat the use of an anionic dendrimer and appropriate choice of linkingchemistry can produce superior therapeutic efficacies, even in cells,without the use of any targeting moieties (Gurdag et al., 2005; Navathet al., 2008).

The PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate can be a very good candidate for invivo testing in neuroinflammation models (Makki et al., 2008). Byachieving a high local drug concentration with conjugates at the targetsite one could overcome the systemic adverse effects of free drug andimprove the therapeutic efficacy significantly with a reduced dose.Recent studies have shown that the PAMAM dendrimers may have anintrinsic ability to selectively accumulate in cells associated withneuroinflammation, upon local or intravenous delivery (Kannan et al.,2007). When this is combined with the lower cytotoxicity and improvedefficacy in the target microglial cells, the potential for superior invivo results could be enhanced. Relative to free drug, this conjugateshows better efficacy compared to ester-linked neutral PAMAMdendrimer-methyl prednisolone conjugate, perhaps due to betterintracellular drug release enabled by the disulfide linker (Khandare etal., 2005).

Conclusions

A PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate has been prepared using a disulfidelinker, that enables relatively rapid intracellular release of the drug.The FITC-labeled anionic dendrimer is rapidly taken up by microglialcells, despite the anionic surface charge. PAMAM-(COOH)₄₆-(NAC)₁₈conjugate is non-toxic even at the higher concentrations tested invitro. PAMAM-(COOH)₄₆-(NAC)₁₈ conjugate is a more effective anti-oxidantand anti-inflammatory agent when compared to free NAC in vitro. Theconjugate showed significant efficacy even at the lowest dose (0.5 mMNAC), where the activity was comparable or better than that of EuniceKennedy Shriver free drug at 8 mM (16× higher dosage). This shows thatdendrimer-NAC conjugates can be effective nanodevices in decreasinginflammation and injury, induced by activated microglial cells indisorders such as cerebral palsy. (Wang, B. et al., Int. J. Pharm.(2009), doi:10.1016/j.ijpharm.2009.04.050.)

Example 19

Poly(Amidoamine) Dendrimer-Drug Conjugates with Disulfide Linkages forIntracellular Drug Delivery

Dendrimers offer well-defined nanoscale architecture, multivalency, andstructural versatility, leading to their emergence as a promising classof nanobiomaterials. One class of the dendrimers that have been widelyinvestigated is poly(amidoamine) (PAMAM) dendrimers. PAMAM dendrimershave been utilized as drug carriers for gene and drug delivery, asantiviral agents and as in vivo imaging agents. When PAMAM dendrimersare used as drug carriers, they can enhance the biodistribution of drugsand possibly take advantage of enhanced permeation and retention effect(EPR) for targeting tumors. Additionally, it was demonstrated that thedendrimer surfaces can be modified with ligands to target specifictissues and tumors, thus capable of active receptor targeting. Forsuccessful clinical applications of dendrimer-drug conjugates to emerge,the dendritic carriers should eventually release the drugs loaded on tothem in a well-defined and favorable rate. The release rates aredependent on the type of linking chemistry used between the drug and itscarriers as well as the nanoscale structure of the dendrimer conjugateand steric effects.

Several dendrimers have been investigated as drug carriers for variouscancer drugs. The conjugates have shown the ability to target tumors andhave led to improved in vivo efficacy. Recent work has shown that theefficacy of anionic PAMAM dendrimer-methotrexate conjugate is betterthan those of cationic conjugates in drug resistant cell lines, perhapsdue to the differences in subcellular distribution and drug release.PAMAM dendrimers were also evaluated as carriers for anti-inflammatoryagents, such as 5-aminosalicylic acid, ibuprofen, naproxen, andmethylprednisolone. These ester or amide-linked conjugates showedimprovements over the free drug and their release profiles were overtimes scales of days to weeks. The intended application in this studyrequires faster release within hours to days based on neonatal rabbitmodels. Recent work on dendrimer-N-acetyl cysteine (NAC) conjugatesshowed significant enhancement in activity over the free drug anddisulfide linkages used have great prospect for delivery of small drugs.Consequently, objective of the work presented here is to determine therelease mechanism and rates of PAMAM-S—S-NAC conjugates in the presenceof various thiol containing species.

NAC is a potent antioxidant as well as mucolytic agent and a precursorof L-cysteine (Cys) and reduced glutathione (GSH). NAC is clinicallyused for reducing neuroinflammation, endothelial dysfunction, fibrosis,invasion, cartilage erosion, acetaminophen detoxification and transplantprolongation. In addition, NAC reduces cellular production ofpro-inflammatory cytokines such as TNF-α and IL-1β. NAC has a low oralbioavailability requiring high doses. When administered intravenously,NAC binds to plasma proteins via covalent disulfide bonds and can alsocause allergic reactions in some patients complicating its use. By usingdrug delivery vehicles such as PAMAM dendrimers, NAC can be protectedfrom protein binding and can be targeted to specific tissues. Theintrinsic ability of PAMAM dendrimers to target neuroinflammation hasbeen shown previously. Therefore, the PAMAM-S—S-NAC dendrimer conjugatescan facilitate in vivo neuroinflammation targeting, combined withenhanced anti-inflammatory and antioxidant effects of NAC, and throughtailored intracellular release.

A key challenge in dendritic drug delivery is release kinetics.Dendrimer drug complexes are shown to be unstable in plasma and buffers.Conjugates with pH responsive linkages are widely investigated but thedifference in pH of biological fluids is usually not very significantespecially through intravenous route. Amide linkages are typically verystable, whereas ester linkages are cleaved faster compared to amides bypH dependent hydrolysis. Hydrazone linkages are more sensitive tochanges in pH compared to ester linkages but the drug and the carriersneed appropriate functional groups to form a hydrazone linkage.Enzymatic release of drugs from higher generation dendrimers has shownto be problematic due to the steric effects and variable enzyme levelsin tissue. Therefore, development of systems that are stable incirculation but rapidly respond to small intracellular molecules fordrug release would make dendrimer conjugates more versatile. One suchcandidate for initiating release of drugs from dendrimers effectively isGlutathione (GSH).

GSH is the most abundant thiol species in the cytoplasm, functioning asa natural oxidant scavenger and the major reducing agent in biochemicalprocesses. The intracellular GSH concentration (2-10 mM) issubstantially higher than extracellular levels (2 mM in plasma), whichprovides opportunities for intracellular delivery of therapeutic agentsby disulfide-linked carriers. Disulfide linkages were utilized onmelamine based dendrimers to incorporate dansyl groups into dendrimerstructure and investigate the disulfide exchange kinetics. Morerecently, photosensitizer mesochlorin (Mce₆) conjugates of linearN-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, linked by disulfidelinkage for photodynamic therapy of cancer treatment was investigated.Various thiol containing species exist (i.e. plasma proteins such asalbumin, lysosomal proteins, etc.) that can induce disulfide exchangereactions. For these purposes, the use of GSH was investigated as wellas other thiol containing species such as albumin (BSA) and cysteine(Cys), for their kinetics of releasing disulfide linked NAC from PAMAMdendrimer conjugates.

Synthesis of PAMAM-S—S-NAC

The scheme for the preparation of the conjugate is outlined in FIG. 57.Briefly, a solution of SPDP in ethanol was added to a solution ofPAMAM-NH₂ dendrimer in PBS (pH 7.4). The reaction mixture was stirred atroom temperature for 2 hours. N-Acetyl cysteine was added to thissolution at once and the reaction mixture was stirred at roomtemperature for 4 hours. The reaction products were diluted with DMSOand dialyzed, first against DMSO followed by PBS, to remove by productsand excess of reactants. The dialysis was then repeated three times (12hours each) with deionized water to remove any salts remaining. Thefinal solution was lyophilized and the purified product was weighted.The overall reaction yield was 71%. The attachment of 16 copies of NACto PAMAM-NH₂-PDP dendrimers was determined by MALDI-TOF and ¹H NMR. From¹H NMR analysis, methyl protons of N-acetyl cysteine are used ascharacteristic peaks. The attachment of NAC to PAMAM-NH₂-PDP dendrimerswas determined by appearance of methyl protons as singlet at 1.94 ppmwhereas attachment of PDP to PAMAM-NH₂ was confirmed by amide protons asmultiplet at 8.40-8.75 ppm. The payload of NAC was calculated by protonintegration method using the amide protons in PAMAM-NH₂ and methylprotons in PAMAM-S—S-NAC. The conjugate payload was confirmed further bythe MALDI peak at 18.3 kDa, that agrees well with molecular masscalculated by ¹H NMR analysis.

Drug Release Studies

Appropriate amounts of PAMAM-S—S-NAC conjugate were dissolved in releasemedia (Citrate or PBS buffers) to form a solution of 1 mg/mlPAMAM-S—S-NAC. One of the thiol containing molecules (GSH, Cys or BSA)was added to the conjugates to form 10 mM, 2 mM, 0.5 mM 0.1 mM or 2 μMoverall thiol group concentrations and to initiate the release of NAC.All samples were run as triplicates for statistical analysis.

As control samples, conjugates were analyzed in both release media inthe absence of reducing agents. The solutions were kept at 37° C. andstirred continuously. At predetermined time intervals, 10 ml of sampleswere withdrawn and immediatelyanalyzed with RP-HPLC and theconcentrations of analytes were determined by using appropriatecalibrations prepared under same conditions.

In Vitro Cytotoxicity Studies

In vitro cytotoxicity of the conjugate, dendrimer and NAC, at conditionssimilar to that used in the efficacy assays, was investigated by MTTassay. Mouse microglial cell line (BV-2) was obtained from Children'sHospital of Michigan Cell Culture Facility. These cells were usedbecause the eventual in vivo applications seek to target microglialcells that become activated as a result of neuroinflammation [33].

To investigate the cytotoxicity of the compounds, the cells were treatedwith the active compound (free drug, dendrimer, or the conjugate) for 3hours. The lipopolysaccharide (LPS) was used to activate the cells forthe ROS assay. For the cytotoxicity study, the LPS treatment wascontinuous for 24 hours. For both groups, the LPS concentration used was100 ng/ml. Three concentrations of NAC were studied: 0.5 mM, 2 mM and 8mM. For the conjugate assays, the concentrations used corresponded toequivalent NAC doses of the free drug treatment groups. Similarly, thedendrimer assays were run by using dendrimer concentrations that wereequivalent to conjugate treatments. Control groups included cellsreceiving only LPS and no other treatment and cells with no LPS or othertreatment. The proportion of viable cells in the treated group wascompared to that of negative control. The cell viability is expressed asmean±SD of three samples per group, and assessed by t-test.

Reactive Oxygen Species (ROS) Assay

Cells were treated with lipopolysaccharide (LPS) to induce theproduction of ROS. The cells were treated with LPS (100 ng/ml) andeither NAC, PAMAM-S—S-NAC, or free PAMAM-NH₂ dendrimers at appropriateconcentrations to study the efficacy of the conjugates for reducing theROS concentrations. In order to quantify the efficacy of conjugates,H₂O₂ released from BV-2 cells (ROS) was measured using the Amplex RedHydrogen Peroxide/Peroxidase Assay Kit using previously establishedprocedures [34]. The data is presented as percent reduction in H₂O₂concentrations in cells treated with the active compound, compared tocells stimulated by LPS but did not receive any treatment. Toinvestigate the effect of treatment time on the efficacy of theconjugates, two sets of experiments were performed: (1) In Group #1, theLPS and the active compounds (free drug, dendrimer, or the conjugate)were added to the cells at t=0, and the efficacy was followed after 24and 72 hours; (2) In Group #2, the cells were treated with the activecompound (free drug, dendrimer, or the conjugate) for just 3 hours,whereas the LPS treatment was continuous for 24 or 72 hours.

Results and Discussions

Synthesis of Conjugates

To facilitate the linking of NAC to dendrimers via disulfide bond aspacer group 3-(2-pyridyldithio)-propanoic acid (PDP) was used. Tointroduce sulfhydryl-reactive groups, PAMAM-NH₂ dendrimers were reactedwith the heterobifunctional cross-linker SPDP. The N-succinimidylactivated ester of SPDP couples to the PAMAM terminal primary amines toyield amide-linked 2-pyridyldithiopropanoyl (PDP) groups. ThePAMAM-NH-PDP synthesized was than reacted with water soluble NAC to getdesired conjugate PAMAM-S—S-NAC. The attachment of NAC to PAMAM-NH-PDPwas determined by the appearance of methyl protons as singlet at 1.94ppm and amide protons as multiplet at 8.40-8.75 in ¹H NMR. Theattachment of multiple copies of NAC to PAMAM-NH-PDP dendrimers wasfurther determined by MALDI-TOF. MALDI-TOF analysis of the unmodifiedPAMAM-G4 dendrimers gave a broad peak at 14.1 kDa, which closely agreesto the theoretical molecular mass of the dendrimers 14.2 kDa.Conjugation of the PAMAM-G4 terminal amine groups to NAC by the linkerresulted in a shift in the mass peak to 18.3 kDa. Each thiopropanoyl-NACgroup has a molecular mass of 250 Da. Therefore, the MALDI data indicatean average of 16 NAC molecules per dendrimer molecule.

TABLE 3 HPLC analysis summary RP-HPLC retention time summary of analytes(min) GSH GSSH NAC GSH-NAC NAC-NAC PAMAM-S-S-NAC 3.8 3.9 4.7 5.3 8.217.4Release Studies

Dendrimer NAC conjugates were analyzed for their drug release mechanismand kinetics in the presence of GSH, Cys and BSA. Buffer solutions withpH 5 (Citrate Buffer) and pH 7.4 (Phosphate Buffered Saline) containingvarious concentrations of these thiol containing moieties were used inrelease studies.

GSH Triggered Release Mechanism and Rates

All GSH concentrations studied were between average plasma (2 μM) andintracellular (2-10 mM) GSH levels. Various GSH concentrations were usedin order to determine the GSH dependent release kinetics of theconjugate. The conjugate solutions contained 730 μM NAC in theconjugated form (1 mg/ml PAMAM-S—S-NAC) at the beginning of the releasestudies. GSH concentration in the release media was compared to theconjugated NAC concentrations in the release solutions for analysis ofthe release kinetics and mechanism. PBS buffer (pH=7.4) was used inorder to demonstrate the GSH dependent release kinetics in intracellularenvironment and in blood. NAC release profile of the conjugate is shownin FIG. 58. In the absence of GSH, the conjugate was stable, and did notrelease any NAC within 3 days.

GSH can reduce the disulfide linkage in the conjugates in two possibleways. The conjugates may release NAC in free form while a GSH attachesonto the dendrimers forming the disulfide bond. The other pathway mayrelease NAC-GSH, leaving the dendrimer with a free thiol group. TheNAC-GSH released can be exchanged again with another GSH molecule andliberate NAC while forming a dimer of glutathione (GSSG). Even thoughdisulfide exchange reactions only transfer the disulfide bond from theconjugate to its dimer form GSSG, slow oxidation reactions can also takeplace forming new disulfide bonds over longer periods of time. For thisreason, NAC-GSH and NAC-NAC was also monitored during the releasestudies.

The results show that the conjugates released significant amounts of NACwithin 1 hour at intracellular GSH concentrations. The release of NACfrom the conjugate in the presence of GSH was fast and near completionwithin 1 hour. PAMAM-S—S-NAC conjugate released 47% of NAC payload infree form and 19% NAC payload in NAC-GSH form, at 1 hour. At highintracellular GSH concentrations NAC-GSH was gradually reduced to freeNAC form at longer times but the overall percentage of NAC released didchange notably. Similar trends were also observed in lower GSHconcentrations. The amount of NAC released from the conjugate did notchange significantly during the time period of 2 hours up to 17 hours(data not shown). This was in agreement with the expected fast disulfideexchange release mechanism. Release mechanism for PAMAM-S—S-NAC in thepresence of GSH is shown in FIG. 59. When the GSH-induced exchangereaction cleaves the disulfide bond on the dendrimer conjugate, NAC canbe released in the free form, with the dendrimer binding the GSH (Route#1). Alternately, NAC can bind GSH to form NAC-GSH (Route 2).Eventually, the presence of the excess GSH allows for the free NAC to bereleased, through subsequent reshuffling reactions.

At intracellular GSH concentrations, the conjugate released 66% and 60%(at 10,000 μM and 2000 μM GSH respectively) of its payload. The extentof release at 10,000 μM GSH solution was only 6% more than the releaseat 2000 μM. The amount of GSH at 10,000 μM and 2000 μM solutions exceedsthe amount of NAC (730 μM) in conjugated form in the release media;therefore the amount released was not affected significantly. On theother hand, when the amount of GSH was limiting (at 500 μM and 100 μMGSH), the NAC release was reduced and was proportional to the GSHavailable; 31% and 6% respectively. When the release studies werecarried out at 2 μM GSH solution, there was no detectable level of NACreleased. The results of release studies at pH 7.4 indicate that theconjugates prepared can release their payload in a very rapid manner inthe presence of GSH. The extent of NAC release will depend on the amountof GSH available, compared to the number of disulfide linkages.

Reducing activity of GSH is attributable to its thiol group. GSH thiolgroup has a pK_(a) of ˜8.8 and its thiolate form is more reactive thanthe thiol form. Therefore pH is an important parameter for reducingactivity of GSH. In order to study the effect of pH on the reducingactivity of GSH, the release studies were repeated at pH 5 (CitrateBuffer). The release at pH 5 was expected to be much slower due to thedifference in thiolate/thiol ratios. The release studies at pH 5 arealso relevant since the dendrimer conjugates are significantly taken upby the cells via endocytosis mechanism and reach the lysosomes where pHis 5. While GSH is not existent in the lysosomes and disulfide exchangereactions are disputed in lysosomes it is believed that other thiolcontaining molecules can carry on the task and take part in disulfideexchange reactions.

The results of release studies with GSH at pH 5 are shown in FIG. 60.The same GSH concentrations were used as the studies at pH 7.4. Therelease profiles clearly indicate that the disulfide exchange reactionwas significantly slowed due to reduced pH. The conjugates releasedtheir NAC payload for extended periods of time up to 20 hours. When theGSH was at intracellular concentrations and in excess of NAC inconjugated form, 95% of NAC payload was released within 20 hours. Therelease rates were slightly faster (nearly completed within 7 hours) at10,000 μM GSH concentration compared to 2000 μM GSH concentration(nearly completed within 20 hours). The completion of NAC release wasapparent from the flattening of the curve on released graph (FIG. 60).The release rates were significantly faster at these intracellularconcentrations compared to lower GSH concentrations studied. At limitingGSH concentrations of 500 μM and 100 μM, the amounts released over 20hours were 58% and 15% respectively. At 2 μM GSH concentration nosignificant amount NAC was released within 20 hours. The conjugatesolutions containing no GSH did not release any NAC within the timeperiod studied. Release studies at pH 5 indicate that, even though thedisulfide exchange reactions are significantly slowed, the conjugatesprepared can provide sustained release of their payload in the presenceof GSH over a period of 20 hours.

It should be noted that the maximum extent of release achieved atintracellular GSH concentrations for the two different pH buffersstudied were slightly different. The conjugate released about 90% of itspayload at pH 5.0 whereas approximately 65% at pH 7.4. This differencemay be caused by free NAC possibly attaching back to its carrier via anoxidation reaction. The oxidation reaction is faster at pH 7.4 comparedto pH 5, therefore possible NAC reattachment may be more significant atpH 7.4. This can limit the equilibrium NAC concentrations to a lowervalue within the release media compared to pH 5. On the other hand, thisshould not be an issue inside the cell, since the reductive environmentis constantly replenished by glutathione reductase enzyme that shouldshift the equilibrium conditions to complete the release process.

Cysteine Triggered Release

The release studies with GSH were repeated with Cys to investigate theability of the amino acid to reduce the conjugate and to compare therates of release to GSH.

The studies were carried out at same thiol group concentrations and inthe same two buffers for investigation of pH effects, discussed earlierfor GSH. The results of NAC release from the conjugate at pH=7.4 and inthe presence of Cys are shown in FIG. 61. The results indicate that Cyswas able to reduce the conjugate and release NAC at slightly fasterrates compared to GSH. The slightly faster release rates can beexplained by lower pK_(a) value of Cys (pK_(a)=8.3) compared to GSH(pK_(a)=8.8).

The extent of release at all Cys concentrations was very similar to theextent of release of the corresponding GSH release studies. Thesimilarity in extent of release combined with the release rates suggeststhat GSH and Cys are not affected by steric hindrance at the dendrimersurface when cleaving the drug from the dendrimer conjugate. While thisshould be obvious when the small size of Cys is considered, it is aquantitative proof that even though GSH is significantly larger comparedto Cys, it is as effective in reducing the conjugate. Thus, GSH can beused as a drug-releasing agent from PAMAM dendrimer conjugates withoutsteric problems most enzymes can face.

The release profile of the conjugate was also determined at pH 5 usingCys as releasing agent as shown in FIG. 62. The rate of release wassignificantly reduced at this pH compared to pH 7.4, consistent with theresult of GSH release studies. The extent of release was limited by theamount of available Cys for reducing the conjugate. The maximum extentof NAC release achieved with Cys for the two pH buffers studied agreedvery well with GSH release studies. At pH 7.4, the release rate was muchfaster but the extent of release was less than the extent of release atpH 5. This suggests that pH of the media not only has directimplications on the rate of drug release, but also on its extent bygoverning the equilibrium concentrations of the thiol species. Cysteineis the most abundant thiol containing moiety in the body and also a partof GSH structure. The release studies with cysteine thus confirm thatCys can also function as a reducing agent for PAMAM-S—S-NAC as well asGSH.

Stability of Conjugates in Bovine Serum Albumin (BSA) Solution

Since the cysteine thiols are active reducing agents like GSH, and theydo release the drug from the conjugate, it may be possible that cysteineresidues on proteins can reduce the disulfide linkages on the conjugatesif they are not sterically blocked. To investigate the reducing activityof Cys in protein structures, bovine serum albumin (BSA) was chosen forrelease studies since it is the most abundant protein in plasma.Additionally, for intravenous administration of conjugates, it isimportant for the drug to stay intact with the carrier until theconjugate reaches its final destination within the body. Thus therelease characteristic in the presence of albumin is crucial forintravenous applications. The release studies in the presence of BSAwere carried out at pH 7.4 and pH 5 as well. The BSA concentrations usedfor the studies were adjusted so that the overall Cys concentrations inBSA release media were the same as the GSH and Cys release studies,discussed earlier. In addition to the five thiol concentrations studied,the stability of the conjugate at plasma BSA concentration was alsoanalyzed.

All of the release studies performed with BSA resulted in no NAC beingreleased from the conjugates over 24 hours. BSA, with ˜67 kDa molecularweight, is much bigger than the conjugate (˜18 kDa). It was evident thatBSA was not effective in reducing the disulfide linkages on theconjugates in any of the concentrations studied, most probably due tosteric effects. This is not surprising since it was previouslydemonstrated that large proteins may have problems as releasing agentsfor dendrimer conjugates. On the other hand, the stability ofPAMAM-S—S-NAC in the presence of BSA solution suggests that theconjugate can protect its payload from premature release while in bloodcirculation.

In Vitro Cytotoxicity

The induction of reactive oxygen species (ROS) production by LPS did nothave significant cytotoxicity compared to control group that did notreceive LPS treatment. The results indicate that there was nocytotoxicity associated with treatment by NAC in any of the MTT assays.When the cells were treated with the mentioned doses of free dendrimeror the PAMAM-S—S-NAC conjugate, the microglial cell viability was betterthan 80% at all doses of the dendrimer and the conjugate. The cells thatreceived 24 hours continuous treatment with dendrimers or the conjugatesshowed some cytotoxicity at the highest dose, whereas the lower dosesdid not produce significant cytotoxicity. For this reason, only thelowest concentration treatment was considered for continuous treatmentefficacy studies. The conjugate was not cytotoxic at the two lowerconcentrations whereas the highest dose generated some cytotoxicity with84% cell survival rate. Similarly, some cytotoxicity associated withfree dendrimer treatment at higher concentrations was observed. Thecytotoxicity of the PAMAM-NH2 dendrimers could be associated to theircationic polyvalent structure. On the other hand, it was apparent thatthe cytotoxicity of the free dendrimers was reduced upon conjugationwith NAC. This is probably due to occupation of the charged surfacegroups of the dendrimer by NAC. It should be pointed out that theefficacy of the conjugate is evaluated only at the lowest concentrationof the conjugate, where the cell survival rate was greater than 95%.

Efficacy Assay (ROS) in Activated Microglial Cells

Reactive oxygen species (ROS) are important oxidative stress markers,and the oxidative stress is usually assessed by measuring ROS levels.Lipopolysaccharide (LPS) treatment is commonly used for activatingmicroglia, resulting in ROS production. NAC reduces the ROS levels dueto its ability to interact with ROS and also its ability to stimulateendogenous GSH synthesis. Suppression of ROS has been used widely toassess the in vivo efficacy of NAC in tissues undergoingneuroinflammatory processes. The reactive oxygen species formed byhydrogen peroxide (H₂O₂) are major contributors to oxidative damage ofneuronal cells and oligodendrocytes in the brain leading to cell deathand brain injury caused by activated microglial cells. Therefore theability of the PAMAM-S—S-NAC conjugate to reduce H₂O₂ levels indicatesthe efficacy of conjugates in the activated microglial cells, which arethe eventual target cells in vivo. Additionally, since the antioxidanteffect of NAC is associated with its thiol group, which is occupied whenin conjugated form, the conjugate would have to release the NAC to haveantioxidant effects. The efficacy of the conjugates is dependent onentry of conjugates into cells and the subsequent release of free NAC.

BV-2 microglial cells were treated with LPS to stimulate ROS productionand increase H₂O₂ concentration. The LPS exposed cells were co-treatedwith saline, NAC, dendrimer or the conjugate simultaneously for 24hours. The results of reduction in H₂O₂ levels when compared to theuntreated control are shown in FIG. 63. NAC treatment resulted in a dosedependant reduction in H₂O₂ concentrations with the lowest dose of 0.5mM showing only 6% reduction in 24 hours and 7% in 72 hours. The highestdose of 8 mM resulted in 57% reduction in 24 hours and 125% reduction in72 hours. When the cells were treated with free dendrimer atconcentrations corresponding to that in the conjugates, there was asmall reduction in H₂O₂ levels, but this effect seemed to diminish overtime as suggested by 35% reduction in 24 hours which decreased to just12% at 72 hours. It is possible that this reduction in H₂O₂ by the freedendrimer may be due to the cationic amine groups on the surface thatmay interact with the peroxide free radicals. The ‘short term’ nature ofthe effect suggests that the charge-balancing mechanism within the cellmay reduce this effect eventually, or it may be possible that at longertime there is not enough dendrimer to ‘reduce’ intracellular H₂O₂ thatis being produced continuously by the cells exposed to LPS. On the otherhand, by conjugation of the linker and the drug, this cationic nature ofthe dendrimer is altered; therefore this effect should be even lesssignificant for the conjugated form of the dendrimer.

When treated with the lowest dose of NAC in the form of a dendrimerconjugate, the efficacy was increased by more than an order of magnitudecompared to free NAC treatment, with 72% reduction in 24 hours and 101%reduction in 72 hours. The efficacy of 0.5 mM NAC equivalent ofconjugate was comparable in efficacy to 8 mM free NAC treatment, whichsuggests that the effective dose of NAC is reduced by about 16 times byadministration in PAMAM-S—S-NAC form. At the 72-hours time point, thecorresponding combined doses of free drug and free dendrimer have asignificantly lower efficacy than the conjugate.

In order to understand the kinetics of dendrimer uptake and thesubsequent intracellular drug release from the conjugates, the LPStreated BV-2 microglial cells were co-treated with NAC, dendrimer orconjugate for only 3 hours, followed by removal and refreshment of cellmedia containing LPS. The cells were then monitored for H₂O₂concentrations for another 72 hours. The reduction of H₂O₂ levels at 24hours and 72 hours after treatment is shown in FIG. 64. Dose dependentefficacy of NAC is observed for the concentration range studied (0.5-8mM). When the cells were treated with equivalent concentrations of NACin conjugated form, there was a significant enhancement in efficacy bothat 24 hours and at 72 hours. Conjugation of NAC to G4-NH₂ dendrimerthrough a fast releasing disulfide linkage has clearly enhanced thecellular entry and activity of NAC. The reduction in effective dose byconjugate treatment is as much as 4-fold at lower drug doses (0.5 mM and2 mM), even with only 3 hours of treatment. While the treatment withfree dendrimers showed some reduction in H₂O₂ levels at 24 hours, thiseffect faded in 72 hours similar to the earlier ROS assay withcontinuous treatment (FIG. 63). Therefore effective intracellulardelivery of NAC by conjugation to G4-NH₂ PAMAM dendrimer is responsiblefor the enhancement in efficacy, especially in the longer time scales.

For both 3-hours and 24-hours treatments, the dendrimer-conjugated NACshows superior efficacy compared to free NAC. The high efficacy ofconjugates even at the lowest dose indicates that the conjugate is ableto release significant amount of its payload intracellularly. This couldbe explained by the fact that the dendrimer may be transporting more ofthe NAC inside the cells, and that the dendrimer conjugate enables asustained delivery of NAC into the activated microglial cellseffectively over several days. After the conjugates are taken up by thecells via endocytosis, they will reside in the lysosomes for a period oftime where the release of NAC may be relatively slow, because of thelower thiol content in the lysosomes. As the conjugate escapes thelysosomal compartment, more NAC will be released at rates determined inthe release studies. The combination of slower intracellular release,and higher NAC uptake enabled by the dendrimer, may be producing alonger therapeutic effect. Therefore, the lysosomal residence times ofthe conjugates may also play a role in determining the time period thatthe conjugate treatment will have efficacy.

For the free drug and the free dendrimer, at the three doses studied,there is a relatively minor difference in the efficacy between 24-hourscontinuous treatment and 3 hours treatment. However, there is asignificant difference in the efficacy of the 0.5 mM conjugate betweencontinuous and 3-hours treatment. This may be explained by an increasein endocytotic uptake due to the ‘activation’ of the microglial cells bysustained LPS treatment. Therefore, as more treatment time is allowed,more conjugate is transported inside the cell, perhaps releasing afactor of 5 or 6 times more drug intracellularly and providing evenhigher efficacy.

Conclusions

A PAMAM dendrimer NAC conjugate which uses a glutathione-sensitivedisulfide linker for intracellular delivery of NAC in neuroinflammationtreatment has been described. The conjugate prepared was characterizedby NMR, MALDI, and HPLC analysis and the NAC payload was found to be14%. Drug release characteristics and mechanism of the conjugate in thepresence of Cys, GSH and BSA in a concentration and pH dependent mannerhas been investigated. The conjugate released significant amounts of NACwithin 1 hour when present at intracellular GSH concentrations and pH.At lysosomal pH, drug release was sustained for about 8 hours. At bothpH buffers, the extent of release was directly proportional to amount offree thiol present. The stability of conjugates against release by largeproteins such as albumin has been demonstrated, which has implicationsfor intravenous conjugate therapies. The cytotoxicity, cellular uptake,and efficacy of the delivery system were investigated in activatedmicroglial cells. The cellular uptake of the dendrimers was relativelyrapid, with significant uptake in the first 4 hours. The conjugateshowed up to an order of magnitude improvement in efficacy of NAC, invitro. The significant improvement in efficacy demonstrates that NAC isbeing effectively transported into the cells and released from itsdendritic carrier in agreement with the release kinetics determined.

PAMAM dendrimer NAC conjugate reported here is a promising deliveryvehicle for NAC, especially when inherent characteristic of PAMAMdendrimers to target neuroinflammation as well as their active andpassive targeting capabilities are considered. This example establishesthat PAMAM dendrimers can release high drug payloads in a short timeintracellularly, through the use of a ‘small’, natural biomolecule GSH.(Kuroglu, Y. E. et al., Biomaterials 30:2112-2121, 2009.)

Example 20

Dendrimer-Drug Conjugates for Tailored Intracellular Drug Release Basedon Glutathione Levels

N-Acetyl cysteine (NAC) is a clinically important antioxidant,antiapoptotic, and anti-inflammatory drug used in the treatment ofneuroinflammation, AIDS, colon cancer, and detoxification of heavymetals (e.g., lead, mercury, arsenic). NAC has been extensively studiedas both a therapeutic agent and direct cysteine precursor. In thetreatment of neuroinflammation, it acts at multiple neuroprotectivesites and has recently been demonstrated to attenuate amniotic andplacental cytokine responses after maternal infection induced bylipopolysaccharide (LPS) and to restore the maternal fetal oxidativebalance and reduce fetal death and preterm birth. Further, a higher doseof NAC remains a primary treatment for acetaminophen overdose andexposure to toxic chemicals and is routinely used in hospitals. However,the use of NAC requires higher and repeated dosing. This is due to thepoor bioavailability and blood stability, caused by the presence of freesulfhydryl groups in NAC, which are capable of spontaneous oxidation andforming disulfide bonds with plasma proteins. Early pharmacokineticstudies have demonstrated low oral bioavailability of NAC (between 6%and 10%), which were attributed to low blood concentrations of NAC. Theneed for high doses can lead to cytotoxicity and side effects, includingincreased blood pressure. NAC is one of the very few drugs beingexplored for treating neuroinflammation in perinatal applications, whereside effects can be very critical.

The design of appropriate dendrimer-NAC conjugates can improve thestability and bioavailability, and at the same time enable intracellularrelease. These are especially important in the eventual interest inperinatal and neonatal applications of dendrimers and NAC. The uniquedesign of conjugates involves linking of the NAC via disulfide bonds tospacer molecules attached to dendrimers. The resulting structure of theconjugates described here achieves two major objectives to ensureefficacy: (a) it may restrict the protein binding of NAC, as the freesulfydryl groups are involved in disulfide linkages; (b) it may enablehigher intracellular levels of NAC and better release of NAC from theconjugate, resulting from disulfide linkages that are cleaved in thepresence of intracellular glutathione (GSH). The results on in vitrorelease and the cellular efficacy toward reducing neuroinflammation inactivated microglial cells shows the improved efficacy of theconjugates.

Over the past few decades, polymeric carriers have been extensivelyexplored for controlled delivery of drugs intracellularly and totargeted tissues. Dendrimers are emerging as a viable class of polymericvehicles (˜5-15 nm) because of the large density of reactive functionalgroups and well-defined structure and monodispersity. This enables ahigh drug payload, but the steric hindrance at the dendrimer surface canmake drug release a challenge when ester or amide linkers are used,especially at higher generations. Active molecules could beencapsulated, complexed, or covalently linked to the polymeric carrier.The polymer can improve the solubility, stability, and blood circulationtimes. Despite several significant achievements of the polymericconjugates, clinical applications still remain elusive, partly due tothe issues of drug release over an appropriate time interval. Commonapproaches in conjugate design involve the use of ester or amidelinkers, which are cleaved hydrolytically or enzymatically. Forpractical applications in drug delivery, increasing the drug payload andengineering the drug release at the appropriate tissue are two keyaspects in the design of polymer conjugates. For intravenousapplications, it is highly desirable to design a linker that is stableduring circulation but enables drug release when it reaches the targetsite. There is a need to design efficient polymer conjugates havingcleavable bonds or linkers, with high drug payloads and appropriaterelease profiles.

Recently, specific chemical reactions, such as the disulfide reduction,have emerged as alternative mechanisms for drug release. Polymericdelivery systems offer an avenue for GSH responsive targeted delivery ofdrugs to tumor tissue. Various carriers such as gold nanoparticles, goldnanorods, mesoporous silica nanorods, nanoparticle inhibitedβ-galactosidase, poly (2-dimethylaminoethyl methacrylate) (PDMAEMA),carbon nanotubes for siRNA delivery, poly (β-amino ester), gelatinnanoparticles, methyl acryloylglycylglycine 4-nitrophenyl ester for DNAdelivery have been used in this regard with reductively cleavabledisulfide spacers. Furthermore disulfide bonds have been incorporated inthe synthesis of cleavable delivery systems for plasmid DNA, antisenseoligonucleotides, peptide nucleic acids, toxins, and anticancer drugs.Therefore, a dendrimer-based delivery system was combined with disulfidechemistry, to develop a GSH-responsive release system with a high drugpayload. The disulfide bonds are easily cleavable, and hence the drugrelease is not compromised. The glutathione (GSH)-mediated release ofbiomolecules from monolayer-protected gold nanoparticle surfaces andmanipulation of their bioactivity in vitro has been demonstrated. GSH isthe most abundant thiol species in the cytoplasm and the major reducingagent in biochemical processes, providing a potential in situ releasingsource in living cells. The intracellular GSH concentration (1-10 mM) issubstantially higher than extracellular levels (0.002 mM in plasma).More importantly, the GSH levels in cancer tissues can be many-foldhigher than those in normal tissues. Therefore, a GSH responsive linkerwill limit plasma release and can promote targeted release.

Experimental Procedure

Detection of Nitrite Production.

The presence of L-PS induces nitrite production, and the subsequentsuppression of this by the dendrimer conjugates is used to assess theefficacy. BV-2 cells (passage 16) were seeded in 24-well plates at10⁵/ml/well and incubated for 24 hours. The medium was removed and 500μL of fresh serum-free medium was added. The cells were exposed to 100ng/mL of lipopolysaccharide (LPS) and various concentrations ofdendrimer conjugates for 3 hours. The medium was removed again, and 500μL of fresh, serum-free medium containing 100 ng/mL of LPS was added.The cells were incubated for 24 and 72 hours, and the culture medium wasremoved for analysis. Control treatments with various concentrations offree NAC, positive controls with LPS induction and no treatment, andnegative controls without any LPS induction were also studied.Accumulation of nitrite in the culture medium was used as a measure ofNO formation. The nitrite concentration was determined by using theGriess reagent system (Cayman) that uses sulfanilamide andN-(1-naphthyl)ethylene diamine. In brief, 100 μL of supernatant fromBV-2 cells exposed to different treatments was incubated with 50 μL ofGriess reagent 1 (sulfanilamide) and 50 μL of Griess reagent 2(N-(1-naphthyl)-ethylenediamine) for 10 min at room temperature. Theabsorbance at 540 nm was then measured, and nitrite concentration wasdetermined using a curve calibrated with nitrite standards.

Intracellular GSH Measurement.

Levels of intracellular reduced glutathione (GSH) was assessedspectrofluorimetrically by monochlobimane staining (13). Briefly, theprocedure for culture and drug treatment was the same as described inthe previous section. Cells seeded in collagen I coated 96-well plateswere washed once with PBS and incubated with 50 uM monochlobimanediluted in phenol red free medium. The fluorescence intensity wasmeasured after 15 min at 37° C. Excitation and emission wavelengths were380 and 485 nm, respectively. Intracellular GSH reduced rate wascalculated according to the following formula: [reduced rate(%)=(fluorescence intensity of EMEM control−fluorescence intensity oftreatment group)/fluorescence intensity of EMEM control×100%].

Results and Discussion

This therapeutic efficacy of polymer conjugates can be enhanced, andside effects reduced, if intracellular drug release can be enhanced.This is especially true in neonatal applications of NAC, where highdoses of NAC are used. To attain this objective, there were developedGSH-responsive dendrimer NAC conjugates incorporating a connectingdisulfide spacer. Use of the disulfide spacer can provide extracellularstability with rapid degradation once internalized in cells, releasingthe free NAC. In the present investigation, there were synthesized andevaluated two dendrimer conjugates, a cationic PAMAM-NH—CO—Pr—S—S-NACand an anionic G3.5-CO-Glutathione-S—S-NAC (G3.5-CO-GS-S-NAC) conjugate,for the first time in dendrimers with a disulfide bond between the drugand the dendrimer through a different spacer. The drug will be releasedat a rate dependent on GSH concentration.

PAMAM-NH—CO—Pr—S—S-NAC Conjugate Synthesis (1).

To conjugate the NAC to dendrimers, the linker SPDP was appended to thedendrimer with the thiopyridine termination. The NAC was covalentlyattached to the PDP linked dendrimer by the formation of disulfidebonds. Synthesis of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)was performed by a two step procedure (FIG. 102). First,3-mercaptopropionic acid was reacted by thiol-disulfide exchange with2,2′-dipyridyl disulfide to give 2-carboxyethyl 2-pyridyl disulfide(FIG. 102, Supporting Information R-V). To facilitate linking ofamine-terminated dendrimers to SPDP, the succinimide group was appendedon SPDP to obtain N-succinimidyl 3-(2-pyridyldithio)propionate (FIG.103, Supporting Information R-VI), by esterification withN-hydroxysuccinimide by using N,N′-dicyclohexylcarbodiimide. Tointroduce sulfhydryl-reactive groups, PAMAM-NH₂ dendrimers were reactedwith the heterobifunctional cross-linker SPDP (FIG. 103, SupportingInformation R-VI). The N-succinimidyl activated ester of SPDP couples tothe terminal primary amines to yield amide-linked2-pyridyldithiopropanoyl (PDP) groups (FIG. 103). After reaction withSPDP, PAMAM-NH-PDP was analyzed using RP-HPLC to determine the extent towhich SPDP had reacted with dendrimers.

The samples were compared to unmodified PAMAM-NH₂ dendrimers. Sampleswere initially run on a linear gradient from 100:0H₂O (0.1 wt %TFA)/acetonitrile to 10:90H₂O (0.1 wt % TFA)/acetonitrile over 32 min.During this gradient, PAMAM-NH₂ was eluted after (13.1) compared to themodified PAMAM-NH-PDP dendrimer. The increased retention time is in linewith the addition of hydrophobic PDP groups. The slight broadening ofthe peaks and appearance of shoulder peaks for both PAMAM dendrimers andPAMAM-NH-PDP might reflect structural defects that occurred duringsynthesis of the dendrimer, for example, by incomplete alkylation of theprimary amines or intramolecular cyclization. The absence ofamine-terminated dendrimer in the pyridyl disulfide-modified dendrimersindicates the completion of the reaction as reflected from the HPLCanalysis. The PAMAM-NH-PDP so obtained was reacted with water solubleNAC to get the desired conjugate. The linking of NAC to dendrimer viathe formation of a disulfide bond was confirmed by HPLC, NMR, andMALDI-TOF (Table 4, Supporting Information R-VII). The NMR and the MALDIdata for the drug payload agree very well with each other, as summarizedin Table 4. The HPLC chromatogram reflected decreased retention time(FIG. 66B) (15.0 min) with the addition of hydrophilic groups. The shiftin retention times for the dendrimer conjugates confirms the conjugationwith NAC; further, the shift to higher retention times indicates thehydrophobic nature imparted due to the spacer molecules and the NAC. Theabsence of the peaks corresponding to NAC and NAC-NAC and SPDP in thechromatogram for the conjugates confirms the purity of the compoundsynthesized.

TABLE 4 Molecular weight estimation (by ¹H-NMR, MALDI-TOF, and ESI-MS)of NAC and PAMAM-NAC conjugates. Molecular weight by HPLC SolubilityGeneration (NMR/MALDI- Pay Purity of elution in no. TOF/ESI-MS) loadconjugate time PBS/H₂O G4-NH₂ 4 14.1 kDa —  100% 14.2 Highly solublePr-NAC — 250 Da  — — — — G4-NH-CO- 4 18.3 kDa 16 99.2% 15.0 HighlyPR-SS-NAC soluble FITC — 389 Da  — 99.5% — Not soluble FITC-G4-NH- 419.0 kDa 18 99.5% 16.0 Highly CO-PR-S-S- soluble NAC- G3.5-COOH   3.511.1 kDa —  100%  8.25 Highly soluble GS-S-NAC —  468 kDa — 99.1%Soluble G3.5-CO-GS-   3.5 19.7 kDa 18 99.5% 12.5 Highly S-NAC solubleThe chromatogram of the PAMAM-NH—CO—Pr—S—S-NAC conjugate showed thepresence of a very small fraction of NAC-NAC as indicated by the slighthump at 8.2 min. Further, the appearance of methyl groups in NMR at 1.94ppm confirms the formation of disulfide bonds between the PAMAM-NHPDPand NAC. The attachment of multiple copies of NAC to PAMAM-NH-PDPdendrimers was determined by MALDI-TOF. Analysis of the unmodified G4dendrimer gave a broad M+ peak at 14.1 kDa (FIG. 65A), which closelycorresponds to the theoretical molecular mass of the dendrimer 14.2 kDa.Coupling of the G4 terminal amine groups with NAC resulted in a shift inthe major peak to 18.3 kDa (FIG. 65B, Table 4, Supporting InformationR-VII). Each thiopropanoyl NAC group has a molecular mass of 250 Da.Therefore, these data indicate an average of 16 NAC molecules perdendrimer molecule (16 thiopropanoyl NAC groups per dendrimer moleculecontaining 64 amine terminal groups, n=3; number of independentexperiments, Table 4, Supporting Information R-VII). ThePAMAM-NH—CO—Pr—S—S-NAC conjugate was tagged with fluorescent dye FITC(FIG. 99) for a cell uptake study. The drug payloads in the conjugateshave been kept moderate, in order to enable high solubilities of theconjugate for in vivo experiments.PAMAM-CO-GS-S-NAC Conjugate Synthesis.

S-(2-Thiopyridyl) glutathione, was prepared from the reaction of2,2′-dithiodipyridine in excess and the corresponding peptide in amixture of methanol and water at room temperature (FIG. 100). Uponcompletion of the reaction, methanol was removed in vacuo and theresidue was washed with dichloromethane. The aqueous solution wassubjected to reverse phase (RP) HPLC purification, and lyophilization ofthe eluent gave the pure product as a white solid (SupportingInformation R-IX). This compound was reacted with NAC in PBS in pH=7.4to get the desired glutathione-N-acetyl cysteine (GS-S-NAC) (FIG. 100)intermediate and purified. The formation of a disulfide bond wasconfirmed by NMR and ESI-MS (Table 4). The appearance of methyl groupsin NMR at 1.90 ppm indicates the formation of disulfide bond between theGSH and NAC. To introduce the GS-S-NAC, PAMAM-COOH was reacted withGS-S-NAC in the presence of PyBop/DIEA to give the desiredPAMAM-CO-GS-S-NAC conjugate (FIG. 100, Supporting Information R-XI).Introduction of S-NAC was confirmed HPLC, NMR, and MALDI. The same typeof MALDI analysis yielded approximately 19.7 kDa (FIG. 67B, SupportingInformation R-X) (18 GS-S-NAC groups for the PAMAM-COOH dendrimers). Thenumber of GS-S-NAC groups was also determined via NMR analysis and theappearance of methyl protons at 1.70 and 1.92 ppm (SupportingInformation R-XI) indicates the formation of GS-NAC conjugate withdendrimer. The NMR and MALDI data for the drug payload agree very wellwith each other, as summarized in Table 4. The yields ofPAMAM-conjugates are high and reproducible.

Release Studies.

The release of NAC from the conjugates was investigated in the presenceof GSH at intracellular and extracellular concentrations. It was assumedthat the release of NAC would occur by the disulfide exchange reaction.GSH and its oxidized form (GSSG) are responsible for forming theintracellular redox buffer. Intracellularly, GSH takes the role ofattacking thiolate moiety and gets oxidized in the process whilecleaving the existing disulfide bonds. Disulfide exchange reactions donot change the total number of disulfide bonds but rather shuffle thespecies forming them. In the present study, the release of NAC from theconjugates by disulfide exchange reaction was confirmed by the HPLCanalysis and is discussed in detail in the following sections.

Free NAC had an elution time of 4.7 min (FIG. 69a ), whereas GSH elutedat 3.8 min (FIG. 69c ). Oxidized forms of NAC and GSH were also analyzedby HPLC and the oxidized form of NAC eluted (NAC-NAC) at 8.2 min (FIG.69b ), while oxidized GSH (GSSG) eluted at 3.9 min (FIG. 69d ). The GSSGpeak was very close to the GSH peak, and when both were injectedtogether, GSSG appeared as a shoulder on the GSH peak (FIG. 69d ). Onthe other hand, NAC-NAC is more hydrophobic than NAC, as indicated bythe higher elution times for the former than NAC in the chromatogram(FIG. 69b ). Hydrophilicity of NAC can be associated with its thiolgroup, and when this group is occupied, the molecule is rendered morehydrophobic, as suggested by the significant increase in its retentiontime when NAC-NAC (FIG. 69b ) was formed. A similar shift to higherretention was observed for GSSG, as indicated by the appearance of ashoulder on the peak seen for retention of GSH (FIG. 69d ). However,this shift in retention time of GSSG is not as significant (FIG. 69d )as that seen for NAC-NAC (FIG. 69b ). The retention time of GS-S-NAC(FIG. 69e ) was 5.3 min, which was longer than both GSH (3.8 min) andNAC (4.7 min). This suggests that occupation of thiol groups reduces thehydrophilicity of both NAC and GSH.

Stability analysis of free NAC and free GSH suggests that both GSH andNAC go through slow oxidation and form their dimers (NAC-NAC and GSSG)by disulfide bond formation when dissolved in PBS. The rate of disulfidebond formation was relatively slow at 25% of NAC and GSH being convertedto their oxidized form over 17 hours. It was determined that, inaddition to their dimers, when NAC and GSH were present together, theyformed GS-S-NAC as well. The formation rates of GS-S-NAC were inagreement with the oxidation rates determined for both NAC and GSHseparately. When NAC and GSH were present together where GSH was inexcess, no detectable NAC-NAC was formed. The conjugates were alsoanalyzed in the absence of GSH to verify the stability of the disulfidelinkage at physiological pH. When the conjugates were placed in PBSbuffer and analyzed for 17 hours, both conjugates were stable and didnot release any of the NAC they carried. The stability of the disulfidelinkage shows that they are capable of carrying their payload withoutany release due to instability in aqueous media at physiological pH. Theextent of the release of drug from both dendrimer-NAC conjugates wasalso analyzed at plasma and intracellular GSH concentrations. Theconjugates and the GSH in required amounts were added to PBS solution,and the solution was analyzed at various time intervals by HPLC for upto 17 hours. UV absorbance peak areas were used to determine theconcentrations of each of the species in the solution, based onappropriate calibration curves. At plasma GSH concentration (2 μM), bothG3.5-CO-GS-S-NAC and G4-NH—CO—Pr—S—S-NAC conjugates were very stable andthey did not release any detectable levels of free NAC within a 17 hoursperiod. For both conjugates, 1% of the NAC payload was found in therelease medium in reduced GS-S-NAC or NAC-NAC forms. The limited releaseof NAC in reduced GS-S-NAC or NAC-NAC forms was very rapid and competedwithin 1 hour. The remaining NAC stayed intact throughout the releasestudy due to depletion of the reduced GSH in the media. This suggeststhat NAC releases from the conjugate rapidly but the amount of NACreleased will be governed by the amount of reduced GSH available.

The expected release mechanisms of both PAMAM-NH—CO—Pr—S—S-NAC andPAMAM-CO-GS-S-NAC conjugates should be similar, with the only differencebeing the linker used. In the presence of excess GSH, the conjugatescontaining disulfide bonds can get shuffled by GSH in two possible ways.The conjugates may release NAC in the free form, while a GSH will attachonto the dendrimers forming the disulfide bond. The other possible wayincludes releasing of GS-S-NAC while the dendrimers have a free thiolgroup. The GS-S-NAC formed can be further shuffled by excess GSH presentand can yield GSSG and NAC. The shuffling reactions will reachequilibrium where the concentration of each species is stabilized. Thesefast shuffling reactions will not change the total number of disulfidelinkages, while slow oxidation reactions can also take place forming newdisulfide bonds.

Both conjugates were analyzed for their release characteristics atintracellular GSH concentration (10 mM). The results suggest that bothconjugates were able to release significant amounts of free NAC withinan hour (FIG. 70). PAMAM-NH—CO—Pr—S—S-NAC conjugate released 47% of theNAC payload in free form within 1 hour. Additionally, 19% of NAC payloadwas found in GS-S-NAC oxidized form. The total NAC that was detachedfrom the dendrimers within 1 hour was 66%. The extent of NAC releaseddid not change significantly after the initial release within 1 hour;41% of NAC payload was found in the free form and 14% was found asGS-S-NAC. The slight decrease in NAC content was most likely due to theerror in concentration determination, and it was within standard errorlimits. Similarly, PAMAM-CO-GS-S-NAC conjugate released 39% of NAC infree form and another 6% in GS-S-NAC form within 1 hour, yielding a 45%total NAC release. At 17 hours, the free NAC content was determined as46%, and 6% of NAC payload was in the GS-S-NAC form. At intracellularGSH concentration, no NACNAC was formed throughout the release studies.Absence of NAC-NAC can be explained by the excess amount of GSH presentin the media, which can transfer the disulfide linkage onto either GSSGor GS-S-NAC by disulfide exchange reactions. After the initial rapidrelease of NAC, the concentrations the cleavage is by fast exchangereactions that reach equilibrium within 1 hour. The difference in drugrelease of the two conjugates may be explained by the different types oflinkers used for attachment of NAC.

The study shows fast release of NAC from the conjugates in intracellularGSH levels and the stability at plasma GSH levels; these results suggestthe similar release mechanism for NAC from the conjugates. This studydemonstrates that PAMAM dendrimer-based NAC delivery systems can bedeveloped for various applications. The above results have significantimplications in designing dendrimer-based drug delivery systems.Enzymatic release of drugs from dendritic delivery systems ischallenging. Smaller generations were shown to be capable of enzymaticcleavage, but lower generations lack the enhancements higher generationshave to offer, whereas higher generations face steric hindrance issues.Commonly used pH-responsive release systems usually provide slower drugrelease over longer time periods unless the release takes place at avery low pH. The two GSH-responsive delivery systems described here havevery fast release kinetics at intracellular conditions and demonstratethat GSH can be used as a reliable releasing agent in dendrimer-baseddelivery systems. Interestingly, the thiol-containing drugs are capableof forming disulfide bonds, and this is one major contributing factorfor their enhanced protein binding and reduced bioavailability. Further,this study shows that the covalent linking of the thiol-containing drugsby disulfide bonds would provide a means of releasing these drugs fromthe carrier systems at the targeted sites.

Efficacy Assay of Conjugates.

NAC exerts its therapeutic effects by decreasing the production ofpro-inflammatory cytokines and reactive oxygen and nitrogen species. Inthe in vivo studies, NAC is used to treat neuroinflammation induced byactivated microglial cells in perinatal brain injury. Therefore, thecellular efficacy of these conjugates was evaluated in the BV-2 mousemicroglial cell line that is activated by LPS. Microglial cellsactivated by LPS release the free radical NO that can result in damageto membranes and DNA of the surrounding cells leading to cell death. Theantioxidative properties of the conjugates were tested by measuring thenitrite levels as a marker of free radical NO production in the cellsupernatant. Free NAC inhibited nitrite production in a dose dependentmanner after 72 hours of incubation. At 24 hours, a time point at whicha relatively lower amount of NO is produced, only the highestconcentration of free NAC (8 mM) inhibited nitrite release. BothPAMAM-NH—CO—Pr—S—S-NAC and PAMAM-CO-GS-S-NAC conjugates showedsignificant inhibition of nitrite production even at the lowestequivalent dose of NAC (0.5 mM). In fact, in both conjugates, withanionic and cationic terminal groups, 0.5 mM NAC in the conjugated formshowed equivalent efficacy to 8 mM of free NAC. The conjugates did notshow a significant dose dependence, at the three concentrationsequivalent to free NAC, perhaps because significant suppression(>60%-80%) was seen even at the lowest concentrations (FIG. 71) for bothconjugates. Perhaps, the use of even lower concentration of conjugatesmay enable us to find dose dependence, but detailed dose dependence isbeyond the scope of this study. At equivalent concentrations, thecationic PAMAM-NH2-NAC conjugate showed slightly better efficacy thanthe anionic PAMAM-COOH-NAC conjugate. From these results, it appearsthat improved intracellular uptake and high drug payload in thedendrimer conjugate may be producing a high local drug concentrationinside the cell to elicit a significant therapeutic response. It alsosuggests that an appreciable amount of the drug is releasedintracellularly even at these relatively short time intervals(especially for polymer conjugates).

The above results have significant implications in both understandingand manipulating drug release mechanisms and achieving controlledintracellular drug release in dendrimer-based delivery systems. Theresults suggest that GSH can be used as a reliable in vivo releasingagent in dendrimer-based delivery systems. As the most abundant thiolspecies in living cells, GSH is the most likely candidate for thedisulfide reduction in previously reported drug delivery systems. Therelatively rapid drug release at intracellular GSH levels is key indendrimer-based conjugates where drug release is typically much slower.The manipulation of GSH concentration in living cells as demonstratedhere conclusively proves that GSH-mediated release is a viable mechanismfor releasing payloads from dendrimer conjugates.

Conclusions

Dendrimer-NAC conjugates were developed as drug delivery systems for thetreatment of neuroinflammation associated with cerebral palsy inperinatal applications. The PAMAM dendrimer-based intracellular drugdelivery system uses a linker that uses GSH as the releasing agent. Twoconjugates were prepared, one based on an anionic PAMAM G3.5-COOHdendrimer and one based on a cationic PAMAM-G4-NH₂ dendrimer. NMR,MALDI, and HPLC showed that the conjugate synthesis was effective andsuccessful. In vitro release studies at different GSH levels have shownthat GSH is responsible for releasing payloads from a dendrimer carrierin buffer. Flow cytometry and confocal microscopy revealed that theconjugates enter the cells rapidly and localize in the cytoplasm. Theefficacy was assessed in activated microglial cells using nitriteinhibition. Both conjugates showed significant efficacy even at druglevels 16 times lower than that of the free drug. These studies addressa key challenge that relates to drug release from polymer in general,and dendrimers in particular. The intrinsic ability of PAMAM dendrimersto target activated microglial cells in animal models ofneuroinflammation. Combined with the findings of these studies, whichallow tailoring of the intracellular release based on glutathionelevels, thus enabling the design of dendrimer-drug conjugates withincreased in vivo efficacy.

Example 21

PAMAM Dendrimers for Brain Delivery of Therapeutics for the Treatment ofCerebral Palsy

Maternal intrauterine inflammation resulting in microglial activationhas been implicated in the development of periventricular leukomalaciaand cerebral palsy. N-acetyl cysteine (NAC) is a drug that is currentlybeing explored for the treatment of neuroinflammation in neonatal andperinatal applications. However, plasma binding of NAC significantlyreduces the bioavailability requiring very high doses (100-300 mg/kg inanimal models). Neutral PAMAM dendrimer-based nanodevices where adisulfide linker is used to link the drug to the dendrimer weredeveloped. This enables tailored intracellular release of the drug in amanner sensitive to the glutathione levels (low in blood circulation,high inside the cells). When this is combined with the ability ofdendrimers to selectively localize in activated microglial cells,significant improvements in vivo performance is achieved. Thenanodevices are evaluated extensively in a rabbit model of cerebralpalsy. The biodistribution and efficacy of intravenously administereddendrimer-drug conjugates are compared with those of the free drug usinga combination of tools. The biodistribution is studied using microPETimaging and tissue confocal microscopy. The efficacy is evaluated usinga combination of neurobehavioral analysis, assessment of brain tissuelevel inflammatory cytokine analysis. The studies show thatdendrimer-drug conjugates are 10-100 times more efficacious that freedrug, suggesting that these conjugates (˜18000 Da) are able to cross theblood brain barrier and deliver the drug significantly better than freedrug.

Example 22

Dendrimer Applications in Maternal-Fetal Medicine

This Example discloses methods and compositions for deliveringtherapeutics to the mother, without affecting the baby, through the useof placental and amniotic sac barrier. The infection can be treated inthe mother, and neuroinflammation treated in the fetus/baby. The Examplealso provides improved detection of cytokines in the amniotic sac, andmultimodal imaging/targeting.

Treatment of neurological disorders historically has been a challengedue to the blood brain barrier (BBB). More than 98% of allsmall-molecule drugs do not cross the BBB. The typical small moleculethreshold is 500 Da. Typical nanoplatforms, such as nanoparticles (100nm), liposomes (˜100 nm), are not expected to cross the BBB.

Biodistribution Results.

Dendrimers preferentially localize in the periventricular region, whereactivated microglia and astrocytes are present, allowing targeting ofneuroinflammatory processes in the brain. Central nervous system (CNS)infections are diseases with high rates of morbidity and mortality.Since the majority of antimicrobial agents discovered so far do notcross the BBB, the treatment of CNS infections is a major binding of NACto plasma proteins reduces the bioavailability significantly (to lessthan 8%). It is highly desirable to have fast intracellular drug releasefrom the dendrimer, since the treatment has to be effective over thefirst few days. If administered IV, the drug has to reach the brain andbe targeted to neuroinflammatory cells. The results disclosed hereinshow that dendrimers can target neuroinflammation. The thiol functionalgroup in NAC is used to create disulfide links that release the drugbased on glutathione levels (intracellular GSH levels are 1000-foldhigher, so minimal release in circulation is expected). Since NAC isconjugated to dendrimers, plasma binding will be minimal.

Dendrimers, for example 5 nm objects, have unique in vivo properties,including targeting neuroinflammation both in the retina and the brain.Taking advantage of the structural and functional aspects of dendrimerscan lead to improved diagnostics and therapeutic applications (nanoscaleeffects in medicine). Upon intravenous administration, dendrimer-NACnanodevices can improve the efficacy by as much as a factor of 100,based on in vivo testing in rabbit models. This is achieved even withoutany targeting moieties on the dendrimers. Dendrimers can thereforefunction as a platform technology (theranostics: therapy anddiagnostics).

N-Acetyl Cysteine (NAC) is a potent antioxidant and anti-inflammatoryagent. NAC is a precursor of L-cysteine (Cys) and glutathione (GSH). NACis used to treat conditions associated with cytoplasmic oxidativestress, such as during inflammation. NAC acts by binding Reactive OxygenSpecies (ROS) and suppresses the production of cytokines such as TNF-αand IL1-β. Clinical uses of NAC include acetaminophen detoxification,stroke, detoxification of heavy metals (e.g. lead, mercury, arsenic), asan antioxidant, and now maternal-fetal applications.

Example 23

Understanding and manipulating the tissue localization and targeting ofnanomaterials in different disease processes is key to improving theirefficacy for specific applications. For example, therapy of severaldebilitating neurodegenerative and neuroinflammatory conditions of thecentral nervous system (CNS) such as hypoxic-ischemic injury, cerebralpalsy, Alzheimer's, multiple sclerosis, and Huntington's disease havenot been feasible due to the inability to deliver adequateconcentrations of the drug into the CNS. Even though intraventriculardelivery of drugs into the cerebrospinal fluid (CSF) results in greaterdrug concentration with a longer half-life in the cerebrospinal fluid(CSF), drug penetration in the parenchyma is limited, with most of thedrug being taken up by the ependymal cells lining the ventricles, ratherthan the target cells. Implants or injections of drugs orconvection-enhanced delivery (CED) into the brain interstitium are othermethods that have been attempted in delivering drugs ornanoparticles/microsomes loaded with drugs into the brain. These methodsare useful for localized areas of injury or disease where diffusion ofthe drug occurs in the area surrounding the site of insertion ordelivery. The drug concentration decreases with increase in the distancefrom the site of injection. Hence these techniques of drug deliverywould be unsuitable for diffuse neuroinflammatory or neurodegenerativedisorders where multiple regions in the brain may be affected. Drugdelivery vehicles that can target the inflammatory cells in targetedareas of the brain.

The unique interactions between dendrimers (with no targeting moieties)and in vivo neuroinflammatory processes are investigated in this study.Inflammatory responses in the brain are associated with the activationof microglial cells, the resident macrophages of CNS that serve the roleof immune surveillance and host defense under normal conditions.Microglial cells are known to be activated by stimuli such as trauma,infection, inflammation and ischemia resulting in the secretion ofpro-inflammatory mediators, generation of reactive oxygen species (ROS)and peroxynitrites that may lead to further neuronal damage. Thedistribution of dendrimers in the presence and absence ofneuroinflammation was studied using a newborn rabbit model of maternalinflammation induced cerebral palsy. There is shown that intrauterineinjection of endotoxin near-term, in pregnant rabbits leads toneuroinflammation as indicated by a robust microglial activation in theperiventricular regions of the brain. This was associated with aphenotype and histologic changes indicating cerebral palsy in thenewborn rabbits. Consequently, delivering anti-inflammatory agents in atargeted manner to activated microglial cells in the central nervoussystem may result in attenuation of the motor deficits and brain injuryseen in cerebral palsy. This strategy will also have broad applicationsin decreasing microglial activation in other neuroinflammatory disorderssuch as Alzheimer's disease, multiple sclerosis and Parkinson's disease.Although in vivo studies have shown that there is very low accumulationof dendrimers in the brain, most of these studies have used normalanimals. The biodistribution of dendrimers appears to be closely relatednot only to its surface moieties which would dictate the interactions ofthe dendrimers with various cells, but also the disease state and invivo conditions that may influence the extent of uptake of thedendrimers by various cells.

The cellular uptake and distribution of fluorescein-labeled neutralpolyamidoamine dendrimers (FITC-G4-OH) was imaged following subduralinjection, in the neonatal rabbit brain with and withoutneuroinflammation. Neutral dendrimers were chosen because of theirimproved biocompatibility, reduced cytotoxicity, and reduced proteininteractions. Moreover, neutral and cationic nanoparticles have beenshown to have the greatest diffusivity in the brain parenchyma whenadministrated by convection-enhanced delivery (CED). In newborns, thecerebrospinal fluid is easily accessible by injection into thesubdural/subarachnoid space through the bregma, which corresponds to theanterior fontanelle or the “soft spot” in human, since the cranialsutures are not completely fused. Local delivery of drugs into the CSFin newborns can be achieved without any more invasive mechanism ofinjection into the interventricular space. The subarachnoid injection ofFITC-G4-OH into the CSF in the newborn rabbit, the tissue images weretaken by fluorescence and confocal microscopy. HPLC analyses ofhomogenized tissue from specific areas of the brain allowed forestimation of dendrimer uptake in the targeted region.

Preparation of FITC-G4-OH Dendrimers

To a solution of fluorescein isothiocyanate (FITC) (80 mg) in anhydrousDMSO, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)(60 mg) and catalytic amount of N-dimethyl amino pyridine (DMAP) wasadded. ^([28]) The reaction was stirred for 20 min and G4-OH (250 mg)was added to it, the reaction was allowed to proceed further for 18hours at room temperature in dark. To remove unreacted FITC and EDC thereaction mixture was dialyzed (molecular cut off of membrane 1000 Da) inDMSO for 24 hours (by changing the DMSO 3 times). The DMSO solvent waslyophilization to get FITC-G4-OH. The FITC-G4-OH compound wasreconstituted into methanol and precipitation in acetone. The productwas dried by lyophilization to obtain FITC-G4-OH (1) conjugates. Absenceof free FITC in the conjugate was verified by TLC using chloroform andmethanol (ratio 1:1) as mobile phase and further by ¹H-NMR and HPLCanalysis. ¹H NMR (DMSO-d₆), δ ppm 2.18 (m, G4-OH protons), 2.39-2.70 (m,G4-OH protons), 3.0-3.16 (m, G4-OH protons), 3.22-3.41 (m, G4-OH)4.65-4.78 (bs, OH protons of G4-OH), 6.56-6.68 (m, aromatic protons ofFITC), 7.76-7.91 (m, amide protons of G4-OH), 6.47-6.59 (d, 6H, Ar,FITC), 6.61-6.72 (s, 3H Ar, FITC) corresponding to the FITC protons andinterior dendrimer amide protons at 7.793-7.63 (br. d, 1H, NH, interiordendrimer amide amide).

Animal Model of Cerebral Palsy

New Zealand White rabbits (Covance Research Products Inc., Kalamazoo,Mich.) with timed pregnancies that were confirmed breeders with ahistory of delivering 7-11 kits per litter, underwent laparotomy undergeneral anesthesia (2-3% isoflurane by mask) on gestational day 28 (E28,term pregnancy is 31-32 days). One mL of saline for the control group(n=3) or 1 mL of saline containing 20 μg/kg of LPS (Escherichia coliserotype O127:B8) (Sigma-Aldrich, St Louis, Mo.) for the endotoxin group(n=3), was equally divided and injected into the uterine wall using a 27gauge needle between the fetuses taking care not to enter the amnioticsac. This ensured that all the kits were exposed to the same amount ofendotoxin. Normothermia was maintained using a water circulatingblanket, and heart rate, oxygen saturations, and arterial blood pressuremeasured through a 20 G arterial catheter placed in the marginal earartery, were monitored continuously during the procedure. Maternal serumwas collected before laparotomy (0 hours) and at 6, 24 hours followingendotoxin injection. The dams were monitored daily for changes inactivity, feeding and fever. A surveillance camera was placed in therabbit room and the dams monitored remotely to determine the time ofdelivery. The kits were born spontaneously at 31 or 32 days gestationalage and the litter size ranged from 7-12 kits, live kits were weighedand recorded.

Tissue Processing

Animals in each group were euthanized 24 hours after subarachnoidadministration of FITC-G4-OH with an overdose of pentobarbital (120mg/kg administered intra-peritoneal). Following administration of thedrug, animals were secured to a stainless steel surgical apparatus, theheart was exposed rapidly and a butterfly needle was inserted andsecured in the left apex of the heart, the vena cava was incised andperfusion was initiated. Animals were perfused under pressure with 30 mLchilled physiological saline (0.9%) and 120 mL of 4% paraformaldehyde inphosphate buffer (0.1 M, pH 7.4) at a constant rate of 5 ml/min using aconstant pressure pump, brains were removed and immersed in the samefixative for 48 hours, cryoprotected using graded solutions of sucroseand frozen at −80° C. until they were sectioned. Brains were embedded in100% OCT media (Tissue-Tek®) and twenty-micron thick coronal brainsections were cut using a cryostat (Leica Microsystems; Nuchloss,Germany) and mounted on poly-L-lysine coated slides (Sigma-Aldrich, StLouis, Mo.).

Lectin, GFAP&MBP Fluorescent-Histochemistry Staining

Brain sections were washed with PBS followed by incubation in 1%hydrogen peroxide for 30 minutes in order to inactivate the endogenousperoxidase, and then in PBS solution containing 0.3% triton X-100 (PBST)and 0.5% bovine serum albumin (BSA) for 1 hours following which slideswere washed thrice with PBS for 5 minutes each. For microglial staining,the slides were covered with Texas red labeled tomato lectin (1:100)(Vector Laboratories, Burlingame, Calif.) overnight. For glialfibrillary acidic protein (GFAP) immunolabeling, brain sections wereincubated with mouse polyclonal GFAP to detect astrocytes (diluted1/500; Dako Cytomation, Glostrup, Denmark). For myelin basic protein(MBP) immunolabeling, brain sections were incubated with rat monoclonalMBP to detect oligodendrocyte (diluted 1/100; Abcam). After over nightincubation, slices were washed with PBS thrice for 5 minutes each andthen incubated with the secondary antibody which wasrhodamine-conjugated goat anti-mouse for GFAP immunostaining (diluted1/500; Abcam.) or rhodamine-conjugated goat anti-rat for MBPimmunostaining (diluted 1/200; Abcam.) for 2 hours. All slides werestained for nuclei using DAPI stain at a concentration of 1 μg/ml for 10mins at room temperature. Slides were then rinsed in PBS, dehydrated ingraded ethanol and cleared in xylene. The slides were then mounted withmounting medium (Sigma-Aldrich) and images obtained using a Leica DM2500microscope (Leica Microsystems; Nuchloss, Germany) equipped with acamera or a confocal microscope (Zeiss LSM 310). The λex=495 nm, λem=521nm for FITC. Injection of equivalent amount of free FITC served ascontrol.

Estimation of FITC-G4-OH in Rabbit Brain Using HPLC.

Brain tissue samples weighing approximately 1 mg were collected fromhealthy pups and CP pups and used for analysis. The tissue sections werehomoginized in 1× ice cold cytoplasmic lysis buffer with manualagitation and repeated for 5 times. The samples were centrifuged at8,000×g for 20 minutes and the supernatant containing the cytosolicportion of the cell lysate were obtained. The samples were analyzed byHPLC and the amount of dendrimer-FITC quantified using the standardcalibration curve for FITC-G4-OH. To estimate the fluorescence as ameasure of concentration of FITC-G4-OH localized in hippocampus orcortex, the samples were analyzed by reverse phase-HPLC and the amountwas quantified using the standard calibration curve for FITC-G4-OH. Allmeasurement were performed in triplicate for statistical analysis.

Results and Discussion

Preparation of FITC-Labeled G4-OH (FITC-G4-OH) Dendrimers

The dendrimer (2) was covalently conjugated to FITC (3) by one-stepsynthesis reaction, through the formation of an ester bond. For this,the selection of an appropriate dendrimer candidate for FITC conjugationis crucial. The higher generation cationic amine-terminated dendrimersare sometimes cytotoxic when compared to the neutral hydroxyl terminateddendrimers. The appropriate dendrimers should have an adequate number ofreactive, surface end groups to conjugate the FITC ensuring optimalpayload. G4-OH dendrimer, which contains 64 hydroxyl groups and is nontoxic within the concentration range used in the present study, wasused. The carboxylic acid group of FITC was conjugated with —OH groupsof PG4-OH dendrimer by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as coupling agent (FIG. 101). Withone-step reaction scheme, reasonable payload of FITC was expectedbecause of the multiple free surface functional groups that areavailable on the periphery of the dendrimer, and the high reactivity ofthe acid group of FITC. The FITC-labeled compound (FITC-G4-OH) (1) waspurified on dialysis using dialysis membrane (cutoff 1000 Da) againstDMSO in dark by replacing DMSO, to remove unreacted compounds. Purity ofFITC-G4-OH conjugate was confirmed by HPLC (FIG. 72) using a florescentdetector (λex=495 nm, λem=521 nm). The FITC-G4-OH conjugate showed asingle peak at 17.5 in the reverse phase HPLC chromatogram indicatingabsence of free FITC which was further confirmed by the comparison ofthe retention times for conjugate and free FITC which were distinct. Theconjugates formed through this condensation reaction were characterizedusing ¹H NMR spectroscopy ¹H-NMR was used to characterize the conjugatebased on the appearance of dendrimer protons at 2.18 (m, G4-OH protons),2.39-2.70 (m, G4-OH protons), 3.0-3.16 (m, G4-OH protons), 3.22-3.41 (m,G4-OH) 4.65-4.78 (bs, OH protons of G4-OH), and aromatic protons at6.47-6.59 (br.d, 6H, Ar), 6.61-6.72 (s, 3H Ar) corresponding to the FITCprotons and interior dendrimer amide protons at 7.793-7.63 (br. d, 1H,NH amide) respectively (FIG. 73). Further ¹H-NMR analysis confirmedattachment of 2 molecules of FITC per dendrimer molecule in FITC-G4-OHconjugate (1).

Subdural Administration of FITC-G4-OH Leads to Localization in ActivatedMicroglia and Astrocytes in Endotoxin Kits with Neuroinflammation.

Pregnant New Zealand White rabbits were injected with endotoxinlipopolysaccharide (LPS) or saline along the length of the uterus at 28days gestation (term gestation=31 days). Rabbit pups that were exposedto maternal endotoxin in utero were born at term spontaneously withcerebral palsy while those that were exposed to maternal salineinjection had a normal phenotype as previously described by the group.The full term pups were born with cerebral palsy (referred to as‘cerebral palsy pups’, n=3) or those born to healthy pregnant rabbitsthat were administered saline (referred to as ‘healthy pups’, controls,n=3) were injected with 2.5 μg of dendrimer-FITC (FITC-G4-OH) in 5 μLPBS into the CSF in the subarachniod space through the skin and dura atthe bregma and sacrificed 24 hours later. Brains were fixed withparaformaldehyde, frozen and sectioned into 20 □m sections, and allsections were examined under fluorescence microscopy for the presence ofFITC-G4-OH. Alternating sections from the para formaldehyde-fixed andfrozen brains were stained for microglia (Texas-red tagged lectin), andfor astrocytes (Rhodamine-labeled Glial fibrillary acidic protein(GFAP))to determine specific cellular co-localization of dendrimer-FITC. In thehealthy pups (control), very little FITC-G4-OH was noted in the brainparenchyma (FIG. 74, healthy pups). Surprisingly, in the pups withcerebral palsy, the distribution of dendrimer-FITC in the brainparenchyma was found to be far-removed from the site of injection andlocalized to the periventricular white matter regions involving thecorpus callosum, internal capsule, along the lateral ventricle andhippocampus, without any uptake noted in the cortex even near the siteof injection (FIG. 74, CP pups). The presence of FITC-labeled dendrimerin these regions was significantly greater in the CP pups withneuroinflammation than in the healthy pups. Based on previous studies,these were the regions that were known to have an increased density ofmicroglia and astrocytes in this rabbit model of CP.

When the microglia were stained with Texas-red tagged lectin, FITC-G4-OHwas found to localize largely in the cytoplasm of activated microglialcells in both CP and healthy kits (FIGS. 75a and 75b ). The activatedmicroglia are recognized by their amoeboid cell body with short andthick processes. Since the CP pups had a significantly greaterexpression of activated microglial cells, there was increased dendrimeruptake noted in these animals. The co-localization of dendrimers andastrocytes were investigated by labeling astrocytes withrhodamine-tagged GFAP. In CP pups, there is significant activation ofastrocytes, indicated by an increase in number, along with theenlargement of the cell bodies and thickening of the processes. Incontrast, in the healthy pups, the astrocytes have thin processes andextensive branching with very small cell bodies. Dendrimer-FITC wasfound to co-localize significantly in activated astrocytes in CP pups(FIG. 76a ), with no co-localization in astrocytes noted in the healthypups (FIG. 76b ).

The increased uptake and specific distribution in the periventricularregions in the CP pups is related to the presence of activated microgliaand astrocytes in these areas. This may be because of the increasedendocytotic ability of activated microglial cells and astrocytes in CPpups with neuroinflammation. Interestingly, cells such asoligodendrocytes and neurons that are typically not involved in causinginflammation do not appear to take up the dendrimers to an appreciableextent (FIG. 77). When equivalent amount of FITC alone was injected intothe CSF, both the CP and healthy pups showed non-specific uptake in alllayers of the cortex and ventricular region (FIG. 78) with relativelyminimal fluorescence seen in the regions associated with inflammatoryactivity where an increased density of activated microglial cells andastrocytes are noted. This suggests that the unique uptake profiledescribed is related to the properties of the dendrimer, rather thanFITC.

Activated microglia and astrocytes, which are the neuroinflammatorycells, are typically found in the periventricular white matter tractsand the hippocampus in the CP pups, with the cortex being relativelyspared of these cells. Localization of dendrimer-FITC in the activatedneuroinflammatory cells would be further confirmed by increased presenceof the dendrimer-FITC in the periventricular regions and the hippocampuswith it being absent in the cortex in CP pups. In order to get asemi-quantitative measurement of the amount of dendrimer-FITC in thedifferent regions of the neonatal rabbit brain, the hippocampus (thatwould be expected to localize dendrimer-FITC in the neuroinflammatorycells) and part of the frontal cortex (that would lack dendrimer-FITCdue to lack of neuroinflammatory cells) were dissected from fiveadjacent 20 μm sections of the brain starting from the level of thebregma in both healthy and CP pups. In both groups there was nofluorescence detected in the cortex indicating that there was nodetectable uptake by cells in the cortex. In the hippocampus, there arenormally a small amount of microglial cells in the control and anabundance of activated microglia and astrocytes in the CP pups. A13-fold greater fluorescence was noted in the CP pups indicatingincreased uptake by neuroinflammatory cells in this region, compared tocontrol (0.0297 μg/mg±0.0044 of FITC-G4-OH in CP pups vs 0.0022μg/mg±0.00095 in healthy pups as detected by HPLC analysis (Table 5).This corresponds well with the histological data where co-localizationof dendrimer-FITC is seen with activated microglia and astrocytes thatare confined to the periventricular white matter regions and thehippocampus with relative sparing of the cortex in the endotoxinanimals. Table 5 summarizes (qualitatively) the regional and cellulardifferences in the biodistribution of the dendrimer in the brain ofhealthy and CP pups.

TABLE 5 Subdural Injection of FITC-G4-OH in Rabbit Model of CerebralPalsy Endotoxin animal Control animal PVR CORTEX PVR CORTEX S. No μg/mgtissue ± SD μg/mg tissue ± SD Pup 1 0.0321 ± 0.0041 Not 0.0015 ± 0.00091Not Pup 2 0.0277 ± 0.0044 detectable 0.0024 ± 0.00098 detectable Pup 30.0292 ± 0.0045 0.0028 ± 0.00096 Average 0.0297 ± 0.0044 0.0022 ±0.00095

When activated with lipopolysaccharide (LPS), microglial cells activelytake up dendrimers with peak intracellular concentrations being achievedwithin 1-2 hours after exposure. Injection of dendrimers into the CSF inthe subarachnoid space (FIG. 79) results in maximal uptake by themeninges and the cells in the cortex since they are most in contact withthe CSF. Instead, it was determined that the dendrimers predominantlylocalize in cells in the periventricular regions and deep within thewhite matter and grey matter regions which are infiltrated withactivated microglia and astrocytes in the newborn rabbits withneuroinflammation. The microglial cells endocytose the FITC-G4-OH fromthe site of injection and migrates to the periventricular region.Microglia constitutes 10% of the total cells in the brain and plays apivotal role in immune surveillance function. Microglia constantlysurvey their local surrounding with their highly motile processes byendocytosing of nutrients and clearing cellular debris. Underpathological condition ramified microglia rapidly transforms intoamoeboid morphology and migrates to the site of injury following achemotaxis signal. In vivo two-photon microscopy demonstrated thatmicroglial cells are capable to migrate within 1-2 days to newly formedamyloid plaques in an animal model of Alzheimer disease (AD). It isplausible that following subdural injection of FITC-G4-OH, microgliamigrates to the site of injection, endocytose FITC-G4-OH and furthermigrates to the periventricular regions. Owing to the highly efficientendocytosis function of microglia, it may be presumed that in thepresence of microglia there may be a limited uptake of FITC-G4-OH byoligodendrocytes or other neuronal cells. Hence, CP pups show higheruptake of FITC-G4-OH into astrocytes and microglia cells compared tohealthy ones indicating a differential uptake of FITC-G4-OH by activatedcells, which is expected due to inflammation.

Conclusion

Understanding the intrinsic targeting potential of nanomaterials (invivo) has a significant impact on the design of targetingnanotherapeutic approaches. Current study suggests endocytosis ofneutral PAMAM-G4 dendrimers in activated microglia and astrocytes thatsubsequently migrate to the location of persistent inflammation (likethe periventricular region) in a rabbit model of cerebral palsy. Theseresults indicate the prospective use of dendrimers as effective drug andgene delivery vehicles, with a potential for targeted therapy forneuroinflammatory conditions such as Alzheimer's, multiple sclerosis,Parkinson's disease and cerebral palsy. The in-vivo results of theselective uptake of PAMAM dendrimer by microgial cells in the rabbitcerebral palsy are further corroborated by the in-vitro results showinguptake of the PAMAM dendrimer in microgial cells.

This shows that nanomaterials injected into the brain can be endocytosedby activated microglial cells and astrocytes and migrate to thepersistent inflammatory region, and may have broad implications in thetreatment of several neuroinflammation-associated diseases, such ascerebral palsy, Alzheimer's, Multiple sclerosis, Parkinson's disease andcerebral palsy in the future.

The most significant finding of the present study is the endocytosis ofPAMAM dendrimer (without a targeting ligand) into the activatedmicroglia and astrocytes. Interestingly, these cells migrate to thepersistent inflammatory region associated with neuroinflammation as seenfrom the immunofluorescence histological evaluation. This shows thatthese dendrimers can be effective drug delivery vehicles to target drugsto CNS for neurodegenerative diseases this study, by establishing that aPAMAM dendrimer (without a targeting ligand) is endocytosed by activatedmicroglia and astrocytes and migrates to the persistent inflammatoryregion associated with neuroinflammation. Thus these dendrimers will beeffective drug delivery vehicles to target drugs to CNS forneurodegenerative diseases.

Example 24

In Vivo Efficacy and Biodistribution of Dendrimer-NAC Conjugates

Results:

5.5 mg/kg of dendrimer-alexa was injected intravenously to both controland endotoxin-administered mothers in the rabbit model. The animals weresacrificed 24 hours later, by administering a cocktail of ketamine andxylazine (IM; 45-75 mg/kg and 5-10 mg/kg respectively). Anesthetizedanimals were secured to a stainless steel surgical apparatus, the heartwas exposed and a butterfly needle was inserted and secured in the leftapex of the heart, the vena cava was incised and perfusion wasinitiated. After blood collection, animals were perfused under pressurewith 30 mL chilled physiological saline (0.9%). After the rabbits weresacrificed, liver, lung, kidneys, large and small intestine, heart,spleen, placenta samples were removed and stored at −80 C until analyseswere performed. The dendrimer was extracted from the thawed samplesaccording to Whelpton's protocol. The extracting solution consisted ofmethanol-dimethyl sulfoxide-water (32:8:1 v/v/v). Tissues were rinsed incold saline, blotted dry on filter paper and weighed. Tissue samples (40mg, depending on the organs) were homogenized 10-20 seconds in 0.1 mLice-cold extracting solution with a homogenizer, repeat this procedure5-8 times. The sample was kept on ice during homogenization. Thehomogenate was then centrifuged (10 min, 600 g) and supernatants werekept for further HPLC analysis. Results on amniotic fluid and placentalsamples collected from an endotoxin-administered rabbit pup and motherrespectively, are shown for the HPLC quantification of dendrimer-alexauptake.

In Vivo Evaluation of Free n-Acetyl Cysteine and Dendrimer-n-AcetylCysteine for the Treatment of Neuroinflammation in a Newborn Rabbit Pupwith Cerebral Palsy

Based on the results that showed that dendrimers can targetneuroinflammation even upon intravenous administration to newborn pupswith CP, the efficacy of dendrimer-NAC in suppression ofneuroinflammation and oxidative stress along with attenuation of motordeficits was evaluated. The neutral dendrimer-NAC conjugate (D-NAC) wasused in this study. Newborn littermate rabbits born with motor deficitssecondary to maternal endotoxin administration were treated with asingle dose of either PBS, NAC 100 mg/kg, D-NAC 1 mg/kg or 10 mg/kg, ordendrimer alone on day 1 of life (31 days post-conception). Newbornrabbits were then subjected to neurobehavioral testing on day 5 of life.Animals were video-taped for 10 minutes and were scored by two observersin a blinded manner. Scores were based on the maximum number of stepstaken without falls (scored from 0-4) and number of hops without falls(scored from 0-4) in one minute of continuous activity. Both scores wereaveraged for obtaining the final score. Since the newborn rabbits arenot able to hop on day 1, the maximum score that can be obtained is 4for day 1 of life and 8 for day 5 of life.

Experimental Design:

Pregnant New Zealand white rabbits at 28 days gestation underwentlaparotomy and endotoxin injections. Controls had no intervention.Littermates were treated at birth with PBS, NAC 100 mg/kg,Dendrimer-NAC(D-NAC) 1 mg/kg, Dendrimer-NAC 10 mg/kg, or Dendrimerlinker.

Significance:

The in vivo results suggest that attenuation of neuroinflammation usingdendrimer-NAC in the newborn leads to significant improvements in themotor deficits, and myelination. When conjugated to the dendrimer, NACis significantly more effective than NAC (by as much as a factor of 100.This is despite the fact that the conjugate (˜18 kDa) was injected IV,and that the dendrimer had no targeting ligands. This validates thedendrimer-based therapeutic approach for neuroinflammation in thismodel, and provides impetus for the proposed future research.

Example 25

Antimicrobial Properties of Neutral PAMAM Dendrimers

Intrauterine infection is usually caused by microorganisms ascendingfrom vaginal and affecting the fetus and amniotic fluid leading tochorioamnionitis, cerebral palsy, increased efficiency of HIVseroconversion, miscarriage, and spontaneous preterm birth (Chaim etal., 1997), (Romero, 2003; Ugwumadu, 2007). Chorioamnionitis is known tocause fetal brain injury (Patrick et al., 2004) due to the generation ofpro-inflammatory cytokines (Dickinson et al., 2009; Harnett et al.,2007). Antibacterial and antifungal agents are applied to vagina andcervix to treat intrauterine infections in the pregnant women (Chaim etal., 1997; Ugwumadu, 2007). E. coli infection in pregnant guinea pig canbe treated by topical vaginal and cervical application of G₄-PAMAM-OHdendrimer. This is the first report using the guinea pig model ofchorioamnionitis to induce E. coli infections and show the effectiveinhibition of bacterial growth by treatment with G₄-PAMAM-OH. Cytokinelevels in placenta of the G₄-PAMAM-OH treated animals were comparable tothose in healthy animals and significantly less than infected animals.Although PAMAM dendrimers are the most extensively studied dendrimersthe antimicrobial activity of unmodified G₄-PAMAM-OH andG_(3.5)-PAMAM-COOH has not been reported previously. Though G₄-PAMAM-NH₂dendrimer shows strong antibacterial activity it shows high cytotoxicityto human cervical cell line and the antibacterial activity ofG4-PAMAM-OH dendrimer is notable since it is non-cytotoxic at higherconcentrations. G₄-PAMAM-OH has a potential as antibacterial agent.

Experimental Section

Materials

The PAMAM dendrimers (generation 4, with end groups OH, NH₂ andgeneration 3.5 COOH 14.93% w/w in methanol) were purchased fromDendritech. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), 2-Nitrophenyl-β-D-galactopyranoside (ONPG), Osmiumtetrozxide N-Phenyl-1-naphthylamine (NPN), Glutaraldehyde andHexamethyldisilazane were purchased from Invitrogen. Nutrient broth andnutrient agar were purchased from BD Biosciences. Mouse TNFα, IL-6 andIL-1β ELISA kits were purchased from R&D Systems.

Preparation of Bacteria

Escherichia coli (ATCC 11775) isolated from human urine is the bacterialstrain used in this study. Single colony on nutrient agar was used toinoculate 5 mL of nutrient broth at 37° C. overnight. A small volume(100 μL) of this growth was used to inoculate 20 mL of nutrient brothmedia at 37° C. for 6 hours. The bacteria were resuspended at 10⁶ colonyforming units (CFU)/mL for the experiments.

Bacterial Growth Inhibition Assays

The inhibitory concentration (IC₅₀) of dendrimers was determined usingthe broth microdilution method (Lopez et al., 2009; Wiegand et al.,2008). Briefly, serial dilutions of dendrimers (0.76 g/mL to 200 mg/mL)were prepared in PBS and combined 1:1 v/v with bacteria at 10⁶ CFU/mL ina 96 well polypropylene plate. After incubation at 37° C. for 18 hours,the absorbance was measured at 650 nm using a microplate reader toassess the cell growth. The positive-control wells contained PBS andnutrient broth medium inoculated with bacteria (5×10⁵ CFU/mL), and thenegative-control wells contained PBS and nutrient broth medium withoutbacteria. The IC₅₀ value was determined as the concentration of thedendrimers which inhibits 50% of microbial growth after 18-24 hoursincubation (Lopez et al., 2009; Wiegand et al., 2008). The % survival ofthe bacteria was determined on the basis of the positive control whichwas considered as 100%.

Evaluation of Normal Cell Cytotoxicity

End1/E6E7 and BV-2 (passage 19) cells were seeded into a 96-well plateat 1.5×10⁴/well, and 5×10³/well, respectively. After 24 hours, cellswere exposed to various concentrations of dendrimers (10 ng/mL to 1mg/mL) in serum free medium for 24 hours. Controls were carried out withmedium alone. Cytotoxic effect was determined using MTT assay. Theproportion of viable cells in the treated group was compared to that ofthe control.

Evaluation the Antimicrobial Activity in Guinea Pig Model ofChorioamnionitis

All the animal experimental procedures were approved by theinstitutional animal care and use committee of Wayne State University.Intracervical bacterial inoculation was performed as previously reported(Patrick et al., 2004). Briefly, pregnant Dunkin-Hartley strain guineapigs (Charles River) at 52 days of gestation were anesthetized with 1.5%isoflurane using the mask. An endoscope was used to visualize thecervix. Guinea pigs were inoculated intra-cervically with 150 CFU E.coli (n=11) to induce infection. Dendrimer G₄-PAMAM-OH 500 μg wasinjected into the cervix 5 min after E. coli inoculation in thetreatment group 3 (n=4). The E. coli inoculated guinea pigs withouttreatment (group 2) were used as positive control (n=4). The guinea pigswithout any treatment (group 1) and inoculation were used as negativecontrols (n=3). Forty eight hours after intervention, guinea pigs wereeuthanized with pentobarbital sodium (120 mg/kg) and midline laparotomywas performed to expose uterus. Amniotic fluid was collected from eachgestational sac and 50 μL was plated on nutrient agar to determine thepresence of microbiologic chorioamnionitis.

Cytokine Quantification in Placenta

The placental tissue (0.3 g) was homogenized in 1 mL RIPA lysis buffer.The homogenate was kept on ice for 30 min, centrifuged at 10,000 g for25 min at 4° C. and the protein concentration of supernatant wasdetermined. Cytokines; tumor necrosis factor (TNFα), interleukin (IL-6and IL-1β) concentrations were measured in the total protein fractionusing ELISA kits (Ethier-Chiasson, 2008).

Statistical Analysis

Data are presented as mean±SD. Specific comparisons between control andindividual experiment were analyzed by ANOVA test with p-value less than0.05 considered as statistically significant.

Results

Antimicrobial Assay

An antibacterial assay procedure reported previously (Lopez et al.,2009) was used to assess the antimicrobial activity of G₄-PAMAM-OH andG_(3.5)-PAMAM-COOH dendrimers towards the gram negative bacteria E. coliand compared it with the activity of G₄-PAMAM-NH_(2.) E. coli was usedin this study since it is known to cause the choriomanionitis conditionin pregnancy and there is an established guinea pig model based on E.coli infection (Patrick et al., 2004). E. coli was used for in-vitro andin-vivo evaluations to demonstrate the antibacterial activity of PAMAMdendrimers. In the present study the IC₅₀ values of PAMAM dendrimerswere measured using a modified broth microdilution assay in a 96-wellplate format. The optical density of the suspension of bacteria indifferent dendrimer solutions was measured at 650 nm. The IC₅₀ value ofthe dendrimer was then obtained from the plot of % survival of bacteriavs. the concentrations of the dendrimer and the plot of opticaldensities vs. the concentrations of the dendrimer. G₄-PAMAM-OH,G_(3.5)-PAMAM-COOH and G₄-PAMAM-NH₂, dendrimers inhibited the growth ofE. coli in a concentration-dependent manner as seen from 18 hourstreatment (FIG. 80B). The strong antimicrobial activity of G₄-PAMAM-NH₂is consistent with that reported previously (Calabretta et al., 2007).It is interesting to note that G₄-PAMAM-OH markedly inhibited the growthof E. coli from 3.13 mg/mL to 25.0 mg/mL concentration.G_(3.5)-PAMAM-COOH also inhibited the growth of E. coli but atrelatively higher concentrations 6.25 mg/mL to 100 mg/mL. The IC₅₀values for G₄-PAMAM-OH, G_(3.5)-PAMAM-COOH and G₄-PAMAM-NH₂ wereobserved as 5.4 mg/mL, 22.0 mg/mL and 3.8 μg/mL respectively. SinceG₄-PAMAM-NH₂ dendrimer exhibits high cytotoxicity, the G₄-PAMAM-OH wasconsidered for in-vivo evaluations in guinea pigs.

For the amine terminated PAMAM dendrimers its proposed that the aminogroups form nanoscale holes in supported lipid bilayers of bacterialmembrane causing its rupture and cell lysis (Calabretta et al., 2007;Hong et al., 2006; Mecke et al., 2005; Milovic et al., 2005). Thequaternary ammonium dendrimers adsorb onto negatively charged bacterialcell surfaces, diffuse through the cell wall, bind to cytoplasmicmembrane, disrupt and disintegrate the cytoplasmic membrane, release ofelectrolytes such as potassium ions and phosphate from the cell andrelease nucleic materials such as DNA and RNA, all contributing to thedeath of the bacterial cell (Chen et al., 2000). These reports suggestthat dendrimers mediate their antimicrobial activity by disrupting thebacterial outer and inner membrane. The antibiotic ampicillin is knownto penetrate the outer membrane of gram negative bacteria and inhibitsthe bacterial cell wall synthesis. The antibacterial activity ofdendrimers is limited to its effect on bacterial membranepermeabilities.

Cytotoxicity Assay

The cytotoxicity of PAMAM dendrimers was evaluated against humancervical epithelial (End1/E6E7) and immune cells; mouse microglial cells(BV-2). MTT assay showed that G₄-PAMAM-OH and G_(3.5)-PAMAM-COOHdendrimers were non cytotoxic to End1/E6E7 cells and BV-2 cells in 24hours treatment at concentrations 10 ng/mL-1 mg/mL (FIG. 85). TheG₄-PAMAM-NH₂ showed high cytotoxicity above 10 μg/mL concentration tohuman cervical epithelial End1/E6E7 cells. Also the G₄-PAMAM-NH₂exhibited cytotoxicity at 1 mg/mL concentration to microglial cells. Onthe basis of the MTT assay, G₄-PAMAM-OH did not exhibit cytotoxicityupto 1 mg/mL concentration, while the G₄-PAMAM-NH₂ was found to becytotoxic at higher concentrations. The indication chorioamnionitis isinduced due to E. coli infections in the vagina. The experimental datashows that G₄-PAMAM-OH dendrimer is non cytotoxic to the human cervicalcell line and also exhibits antibacterial activity towards E. coli,hence it was chosen as antibacterial agent to treat chorioamnionitis inpregnant guinea pigs. In the in-vivo experiments a total of 500 μg ofG₄-PAMAM-OH were applied to the cervix of E. coli infected pregnantguinea pigs and at this concentration the dendrimer showed efficacy.

Antimicrobial Activity in Guinea Pig Model of Chorioamnionitis

The in-vitro studies brought out antibacterial potential of G₄-PAMAM-OHdendrimer as seen from the antibacterial assay, OM and IMpermeabilization assays and bulk changes in morphology seen from SEManalysis. These interesting results coupled with its non cytotoxicity tohuman cervical epithelial cells encouraged the evaluation of G₄-PAMAM-OHas an antibacterial agent in-vivo using the guinea pig model ofchorioamnionitis. Though this model is established for creatinginfection and assessing injury to the fetus, it has not been previouslyused to demonstrate the effective treatment. This shows the treatment ofthe pregnant guinea pig using the model of chorioamnionitis. Theascending E. coli infection causes chorioamnionitis which is associatedwith development of cerebral palsy, a motor disorder in children due tostimulation of proinflammatory cytokines causing white matter damage andfetal brain injury (Patrick et al., 2004). The dose of the E. coliinoculation in the guinea pigs (n=17) was optimized in the pilotexperiments for the strain (ATCC 11775). 1000 CFU of E. coli effectivelyinduced the infection causing extreme sickness in mother and furtherthis dose lead to abortion of dead fetuses within 48 to 72 hours. Thelower CFU of E. coli were subsequently inoculated to identify theoptimum dose, which lead to infection and yet the guinea pigs did notabort upto 48 hours. Based on this evaluation a dose of 150 CFU of E.coli was found to effectively induce the infection in the pregnantguinea pigs without leading to abortion of fetuses.

In the present study, chorioamnionitis was induced after intracervicalinoculation with E. coli in 8 guinea pigs of which n=4 were consideredas positive control (group 2) and n=4 were used for treatment withG₄-PAMAM-OH (group 3). None of the amniotic fluid samples plated fromthe negative control group-1 (n=3) showed evidence of microbiologicchorioamnionitis. Of the pregnant guinea pigs (group 2, n=4) that wereinoculated with 150 CFU of E. coli, 57.1% of the amniotic fluid samplesfor different fetus were positive with bacterial growth (indicative ofinduced infection) as seen from the culture inoculated with it (seeTable 6). Prenatal exposure to maternal infection alters cytokineexpression in the placenta (Urakuboa, 2001). Abundance of cytokines inplacental tissues is an indicator of activation of inflammatory responsein gestational membranes with term and preterm parturition (Keelan etal., 1999). The cytokine IL-6 is known to peak after 48 hours ofinfection (Dickinson et al., 2009) and hence in present study animalswere sacrificed after 48 hours to determine the cytokine level inpositive and treated animals. When the expression of cytokine levels innegative control vs the positive controls were compared, the cytokinesespecially TNFα and IL-6 increased significantly in placenta of positivecontrols after 48 hours of inoculation with E. coli (FIG. 87). Theseresults demonstrated that chorioamnionitis was successfully inducedafter 48 hours of cervical inoculation with 150 CFU of E. coli.

The G₄-PAMAM-OH dendrimer was applied topically at a dose of (625 μg/kg)on the cervical endometrium of guinea pigs (group-3, n=4) in form ofaqueous solution in saline after E. coli inoculation. The total amountapplied was 500 μg dissolved in saline based on the average weight ofthe guinea pigs (800 gm/animal). The amniotic fluid samples fordifferent fetus were collected after 48 hours and were plated on theculture plates and evaluated for the bacterial growth. All these samplesdid not show any bacterial growth (0%) on the culture plates (Table 6).The study shows that the treatment with G₄-PAMAM-OH dendrimer completelyeliminated the bacterial growth and prevented bacteria ascension intouterine cavity and amniotic fluid i.e. from 57.1 (positive control) to0% (treatment group) bacterial growth. Earlier, the in-vitro data showedantibacterial nature of G₄-PAMAM-OH at higher concentration and thein-vivo results show that amniotic fluid samples for different fetus intreatment group-3 were found to be negative. The comparison of cytokineexpression in placenta of the treatment group, negative and positivecontrol groups shows that the cytokine levels (TNFα and IL-6) intreatment group are comparable to the negative control while they areoverly expressed in positive controls (FIG. 87). These results indicatethe potential of G₄-PAMAM-OH to effectively kill gram negative bacteriaE. coli in cervix of guinea pig and prevent chorioamnionitis. This is asignificant finding since the chorioamnionitis is known to cause fetalbrain injury (Patrick et al., 2004) which could possibly be averted bytreatment with G₄-PAMAM-OH as indicated from these findings.

Conclusion

The bactericidal activity of hydroxyl and carboxylic acid terminatedPAMAM dendrimer was evaluated against gram negative E. coli and comparedwith amine terminated PAMAM dendrimers. The antimicrobial assay, SEManalysis, cell integrity, inner and outer membrane permeability assaysshowed that G4-PAMAM-OH and G3.5-PAMAM-COOH dendrimers affect the cellwall of E. coli, and were antibacterial at the concentrations evaluated.The major finding was the bactericidal effect of G₄-PAMAM-OH dendrimerand its ability to treat E. coli infections in-vivo in pregnant guineapigs. Topical cervical application of 500 μg of G₄-PAMAM-OH treated theE. coli infections induced in guinea pig model of chorioamnionitis. Theamniotic fluid collected from different fetus in the infected guineapigs, post treatment showed absence of E. coli growth in the culturesplated with it. The cytokines levels were higher in the positivecontrols confirming presence of infection after inoculation with E.coli. The cytokine expression (TNFα and IL-6) in the treatment group wascomparable to that in negative control showing the efficacy ofG₄-PAMAM-OH to treat the E. coli infections. The G4-PAMAM-NH₂ dendrimeris known to be potent antibacterial agent, however, it was found to behighly cytotoxic to above 10 μg/mL to human cervical epithelial(End1/E6E7) cells and immune cells (BV-2) while the G₄-PAMAM-OHdendrimer was non cytotoxic upto 1 mg/mL concentrations to both celllines. Each dendrimer appears to affect the bacterial cell wall in adifferent way. The possible mechanisms involve the G₄-PAMAM-NH₂ actingas polycation binding to the polyanionic lipopolysaccharide, theG₄-PAMAM-OH binding via hydrogen bonds to the hydrophilic O-antigens andthe G_(3.5)-PAMAM-COOH acting as a polyanion chelating the divalent ionsin outer cell membrane. The outer and inner membrane permeabilizationassay shows that G₄-PAMAM-OH brings major structural changes to theouter membrane whereas G₄-PAMAM-NH₂ brings major changes to both outerand inner membrane.

TABLE 6 The inhibition of E. coli growth after treatment withG₄-PAMAM-OH dendrimer in guinea pig model of chorioamnionitis Treatment:With G4-OH after Inoculation with E. coli E. coli inoculation Amnioticfluid from Amniotic fluid from different gestational differentgestational Guinea sacs Guinea sacs Pig Bacterial growth % Pig Bacterialgrowth % Mother 1 Fetuses (4/5) 80.0 Mother 1 Fetuses (0/5) 0 Mother 2Fetuses (5/6) 83.3 Mother 2 Fetuses (0/6) 0 Mother 3 Fetuses (1/4) 25.0Mother 3 Fetuses (0/5) 0 Mother 4 Fetuses (2/5) 40.0 Mother 4 Fetuses(0/3) 0 Average 57.1 Average 0

Example 26

Dendrimers can Provide Selective Treatment to Pregnant Women, withoutAffecting the Fetus

Synthesis of G₄-PAMAM-O-GABA-NHBoc

The solution of Boc-GABA-OH (914 mg, 4.50 mmol) in DMSO/DMF (3:1) wascooled to 0° C. and then added to the solution of EDC (860 mg, 4.50mmol), DMAP (549 mg, 4.5 mmol) and G₄-PAMAM-OH (1000 mg, 0.070 mmol) inDMSO/DMF (3:1). The reaction mixture was stirred at room temperature for24 hours. The reaction mixture was purified by dialysis with DMSO (24hours) to remove by-products and the excess of reactants. After dialysisthe solvent was removed under lyophilization to get pure compound in 78%yield (889 mg, 0.055 mmol). ¹H-NMR (DMSO-d₆, 400 MHz), δ (in ppm): 1.37(s, 9H), 1.50-1.66 (m, 2H) 2.10-2.20 (m, 4H), 3.97-4.03 (br s, 1H),6.77-6.85 (br s, NH amide from GABA-NH-Boc), 7.70-8.05 (3 br s, NH amidefrom interior of dendrimer).

Synthesis of G₄-PAMAM-O-GABA-NH₂

G₄-PAMAM-O-GABA-NHBoc (1.0 g, 0.062 mmol) was treated withtrifloroacetic acid and dichloromethane (1:1, 10 mL). The reaction wasstirred at room temperature for 10 min. After completion of thereaction, trifloroacetic acid/dichloromethane were removed under vacuumusing rotavapor equipped with NaOH trap. Reaction mixture wasneutralized with Na₂CO₃ and dialyzed with water (12 hours) and solventwas removed under lyophilization to get pure compound in 92% yield (861mg, 0.057 mmol). ¹H-NMR (DMSO-d₆, 400 MHz), δ (in ppm): 1.65-1.78 (m,2H), 2.2-2.39 (m, 4H), 3.91-3.99 (br s, 1H), 7.8-7.98 (br d, NH amidefrom GABA-NH₂), 8.03-8.25 (br d, NH amide from interior of dendrimer)

Synthesis of G₄-PAMAM-O-GABA-NH-FITC

To a solution of G₄-PAMAM-O-GABA-NH₂ (2.50 g, 0.167 mmol) in anhydrousDMSO (50 mL) was added fluorescein isothiocynate (FITC) (800 mg, 2.05mmol) and stirred. The reaction was allowed to proceed further for 18hours at room temperature in dark. To remove unreacted FITC the reactionmixture was dialyzed (molecular cut off of the membrane is 1000 Da) inDMSO for 24 hours. The compound was dissolved in methanol andprecipitated in acetone. The product was dried by lyophilization toobtain G₄-PAMAM-O-GABA-NH-FITC conjugate in the 75% yield (1.98 g, 0.125mmol) and analyzed by ¹H-NMR and MALDI TOF/MS. Absence of free FITC inthe conjugate was verified by TLC using chloroform and methanol (ratio1:1) as mobile phase and further by HPLC analysis.

Synthesis of G₄-PAMAM-O-GABA-NH-Alexa-488

Alexa Fluor 488 carboxylic acid, succinimidyl ester (2 mg, 0.0013 mmol)was added to a solution of G₄-PAMAM-O-GABA-NH₂ (17.5 mg) in PBS (pH=8)(3 mL) and the reaction was stirred at room temperature in dark for 15hours. The reaction mixture was dialyzed in DMSO (molecular cut off ofthe membrane is 1000 Da) for 24 hours in dark. The product was dried bylyophilization to obtain G₄-PAMAM-O-GABA-NH-Alexa conjugate in the yield78% (1.67 mg, 0.0001 mmol) and analyzed by MALDI TOF/MS. Absence of freeAlexa in the conjugate was verified by TLC using chloroform and methanol(ratio 1:1) as mobile phase and further by HPLC analysis.

Dynamic Light Scattering Measurements

Dynamic light scattering (DLS) analyses were performed using a MalvernInstruments Zetasizer Nano ZEN3600 instrument (Westborough, Mass.) withreproducibility being verified by collection and comparison ofsequential measurements. G4-PAMAM-O-GABA-NH-FITC andG4-PAMAM-O-GABA-NH-Alexa conjugate samples were prepared using PBSpH=7.4. DLS measurements were performed at a 90° scattering angle at 37°C. Z-average sizes of three sequential measurements were collected andanalyzed.

Chorioamniotic Membrane Specimens

Study Design

All the human fetal (chorioamniotic) membrane samples were collectedfrom women in uncomplicated normal pregnancies, immediately afterelective caesarian section performed prior to the onset of labor. Fetalmembrane samples were obtained from 21 normal pregnancies from the bankof biological samples of the Perinatology Research Branch.

Chorioamniotic Membrane Processing

All human fetal membrane specimens were obtained at the time of cesareansection for obstetrical indications. The fetal membrane comprisingchorioamniotic membrane (intact membranous tissue containing both amnionand chorion together) (n=7) and amnion (n=7) and chorion (n=7) wereseparately procured (size 6×9″). Each membrane was cut into 9 pieces forthe 9 sets of diffusion chamber. The fresh membranes were collectedimmediately after the delivery, washed with PBS to remove the blood andstored in PBS until (˜1 h at 4° C.) it was mounted on the diffusionchamber (37±0.5° C.). Excess membranes were trimmed. The diffusionexperiments were performed for 48 hours by mounting the membrane in thechamber to study the transport across the membranes. In all theexperiments with chorioamniotic membrane the choriodecidua (maternalside) was placed facing the donor chamber and the amnionic epithelium(fetal side) was facing the receptor chamber. The chorion and amnionwere mechanically separated by gently pulling the two membranes apart.For the chorion, the side attached to amnion was placed facing thereceptor chamber and for the amnion the side attached to the chorion wasplaced facing the donor chamber in the diffusion apparatus. Forhistological evaluation, the tissue samples were collected at 0.5, 1, 2,3 and 4 hours for and were fixed in 10% formalin overnight. Further,some sections were analyzed by hematoxylin and eosin (H & E) staining.The membrane thickness was measured using the Mitutoyo Super Caliper byplacing the respective membranes between the two glass slides andsubtracting the thickness of the glass slides without the membrane.

Immunofluorescence

An immunofluorescence study was performed to investigate biodistributionof the dendrimer through the different layers of the fetal membrane withprogression of time. The fetal membrane tissues were removed from theside by side diffusion chambers at different time points and fixed in10% formalin overnight. Double immunofluorescent staining was performedon 5 μm thick, paraffin sections of membranes placed on silanizedslides. The different regions in the fetal membranes were identifiedbased on the presence of trophoblast in the chorion as documented bystaining with cytokeratin and the presence of the stromal cells asdocumented by staining with vimentin. The immunofluorescent staining wasperformed using Ventana Discovery autostainer for controlled andoptimised reaction environment using the automation-optimized reagentsfrom Ventana Medical Systems Inc. Briefly, paraffin wax sections wereloaded onto the Ventana Discovery platform and following steps werecompleted automatically, these included dewaxing by EZ prep buffer(Ventana Medical Inc.), pre-treatment in Tris/EDTA pH 8.0 antigenretrieval solution (Ventana mCC1) or protease solution for 1 hour(Ventana protease 2). Endogenous peroxidase was inactivated using anenhanced inhibitor provided in the staining kit and nonspecific antibodybinding was blocked by treatment with blocking solution for 10 min. Theblocking solution was removed and the sections were washed three timeswith PBS/Tween solution incubated with primary antibodies for 1 hourusing the liquid cover slip (Ventana Medical Inc). The primaryantibodies used were monoclonal mouse anti-human cytokeratin (1:200,M7018, Dako Carpinteria, Calif., USA) and rabbit polyclonal IgG vimentin(H-84) (1:100, sc5565, Santa Cruz Biotechnology Inc). The sections wereagain washed three times with PBS/Tween solution incubated withsecondary antibodies, Alexa Fluor®594 goat anti-mouse IgG (1:500,A11005, Invitrogen) and Alexa Fluor® 633 F(ab′)₂ fragment of goatanti-rabbit IgG (1:500, A21072, Invitrogen) for 1 hours using theantibody diluent from Ventana. The sections were washed with PBS/Tween,counterstained and mounted with DAPI prolong Gold antifade and coverslipped. Negative controls replaced primary antibodies with rabbitisotype control and mouse isotype controls (Invitrogen) in PBS. Imageswere captured from Leica TCS SP5 Laser Scanning Confocal Microscope(Leica Microsystems GmbH, Wetzlar, Germany). All study specimens wereanalyzed by a pathologist blinded to the clinical information.

In Vitro Permeability Study

Permeation experiments were carried out using a two-chamber (donor andreceptor) side-by-side Permegear diffusion cell with a chamber volume of3 mL and with a diameter of 13 mm and a diffusional area of 1.32 cm².The fetal membranes (chorion and amnion together, amnion and chorion)each (n=9) were mounted between two halves of the donor and receptorcell (9 sets), which were further clamped together and sealed tightlywith the rubber packing at the end of each glass chamber. The receptorcell (volume 3 mL) was filled with sterile PBS (pH 7.4). The donor cell(volume 3 mL) was filled with solution of compounds whose permeabilitywas evaluated. The solutes chosen for the permeation study were FITC(MW=389 Da), G₄-PAMAM dendrimers (G₄-PAMAM-O-GABA-Alexa (2), Mw=˜16 kDaand G₄-PAMAM-O-GABA-FITC (1), Mw=˜15.8 kDa). The system was maintainedat 37° C. by using a circulating water bath and a jacket surroundingeach cell. The donor and receiving medium was continuously stirred (600rpm) with a magnetic bar to avoid stagnant aqueous diffusion layereffects. Aliquots (200 μL) were collected from the receptor cell every30 min till first 6 hours and at predetermined intervals thereafter andreplaced with equal volume of PBS to maintain sink conditions throughoutthe study. The concentration of the solutes used wereG₄-PAMAM-O-GABA-Alexa (0.6 mg/mL), G₄-PAMAM-O-GABA-FITC (3 mg/mL and 0.6mg/mL), and FITC (0.3 mg/mL). The concentration of compound in thereceptor medium was determined using a Molecular Devices SpectroMax M2,UV visible and Fluorescent plate reader at ex 495/em 521. The cumulativeamount of compound transported across the membrane in the receptor cellwas determined using a calibration curve (a transport of 50% correspondsequilibrium achieved). All the experiments were conducted in dark room.All experiments were done in triplicate and the results are reported asmean±STDev.

Results and Discussion

A variety of in-vitro approaches have been used to assess the transportand permeation characteristics of drugs administered by differentroutes, such as permeability across the skin for topical formulationsand permeability across the intestine, colon and jejunum for orallyadministered drugs. Recently, dendrimers have been considered fortopical applications to the vaginal and cervical mucosa asantimicrobicidal agents. The fetus is separated from the extra-amnioticspace in the uterus by the fetal membranes and the ascending genitalinfections in pregnant women are treated by topical intravaginalapplication of antibacterial and antifungal drugs. Since the dendrimersare explored as topical antimicrobial agents themselves and also ascomponents of topical gel formulations, the transport, permeation andbiodistribution of dendrimer across the human fetal membranes wasevaluated in the present study. The selective treatment for the motherwithout affecting the baby is always desired. The present study differsfrom the transplacental transport, where the transport of molecules ordrugs across the placenta is evaluated and is relevant for substancespresent in systemic circulation of mother following administration byoral, parenteral or any other route. The purpose of the present study isto determine whether (a) dendrimers on topical application to vagina andextra-amniotic cavity in pregnant women cross the fetal membrane and (b)could the dendrimers be used for site specific (local) activity and ascomponents of topical delivery systems in pregnant women withoutcrossing the fetal membranes and affecting the fetus.

Preparation of FITC-Labeled G₄-PAMAM-O-GABA-NH₂ Dendrimer (1)

To conjugate FITC to G₄-PAMAM dendrimer with hydroxyl terminations, thelinker GABA with the amine groups protected with Boc(tert-butoxycarbonyl) was appended to the dendrimer to yieldG₄-PAMAM-O-GABA-NHBoc. First, 4-(tert-Butoxycarbonylamino) butyric acidwas reacted with G₄-PAMAM-OH to give G₄-PAMAM-O-GABA-NHBoc (FIG. 104)and the product so obtained was purified by dialysis using DMSO (cutoff1000 Da). The ¹H NMR spectrum shows the appearance of characteristicsignals of G₄-PAMAM-O-GABA-NHBoc at 1.37 (s, 9H), 1.50-1.66 (m, 2H)2.10-2.20 (m, 4H), 3.97-4.03 (br s, 1H), 6.77-6.85 (br s, NH amide fromGABA-NHBoc), 7.70-8.05 (3 br s, NH amide from interior of dendrimer).This confirms the formation of G₄-PAMAM-O-GABA-Boc product. It isevident from the integral ratio of the amide protons ofG₄-PAMAM-O-GABA-NHBoc at 7.70-8.05 ppm to the four methylene protons ofGABA at 2.10-2.20 (m, 4H), that each G₄-PAMAM-OH dendrimer containsapproximately 10 copies of GABA-NHBoc molecules attached to it. Themolecular weight of GABA-NHBoc is 203 Da and the increment in mass ofG₄-PAMAM dendrimer (from ˜14 kDa) to 15960 Da as observed from MALDI TOFMS analysis further confirms the attachment of approximately 10 copiesof GABA-NHBoc to the dendrimer (FIG. 88). The product so obtained wasdeprotected to remove tert-butoxycarbonyl groups by treatment withtrifloroacetic acid in dichloromethane to obtain the amine-terminatedG₄-PAMAM-O-GABA-NH₂ dendrimer. ¹H NMR spectrum shows the disappearanceof characteristic signals at 1.37 ppm corresponding totert-butoxycarbonyl after the deprotection step. Further, the spectrumshows presence of methylene protons at 1.65-1.78 (m, 2H) 2.2-2.39 (m,4H) and amide protons at 7.8-7.98 (br d, NH amide from GABA-NH₂),8.03-8.25 (br d, NH amide from interior of dendrimer) confirming thedesired product G₄-PAMAM-O-GABA-NH₂. The MALDI-TOF/MS analysis ofG₄-PAMAM-O-GABA-NH₂ shows mass corresponding to 14,949 Da (FIG. 88). Themass of GABA is 103 Da suggesting an attachment of 10 molecules of GABAon G₄-PAMAM-O-GABA-NH₂ (MALDI showed mass of G₄-PAMAM-OH as ˜14 kDa).

The G₄-PAMAM-O-GABA-NH₂ dendrimer was tagged with fluorescent dye FITCas shown in FIG. 104. The FITC-labeled compound(G₄-PAMAM-O-GABA-NH-FITC) was purified by dialysis (membrane cutoff 1000Da) against DMSO in dark by replacing DMSO, to remove un-reactedcompounds. Purity of G₄-PAMAM-O-GABA-NH-FITC conjugate was confirmed byHPLC using fluorescent detector (λex=495 nm/λem=521 nm). TheG₄-PAMAM-O-GABA-NH-FITC conjugate showed a single peak at 17.5 min inthe reverse phase HPLC chromatogram indicating absence of free FITC inthe conjugate after dialysis (FIG. 89). The stability of the conjugatein PBS (pH 7.4) after 72 hours was evaluated by HPLC analysis whichshowed a single peak for the conjugate and FITC was not released fromthe conjugate. This observation is consistent with previous reportswhere drugs conjugated to dendrimers by amide linkage are not releasedby hydrolytic or enzymatic degradation. ¹H-NMR was used to characterizethe conjugate. ¹H-NMR spectrum shows the appearance of aromatic protonsat 6.47-6.59 (d, 6H, Ar) and 6.61-6.72 (s, 3H Ar) corresponding to theFITC protons confirming the tagging of FITC on G₄-PAMAM-O-GABA-NH₂. TheMALDI TOF/MS spectrum showed that the mass of G₄-PAMAM-GABA-NH₂increased from 14,949 Da to 15,805 Da suggesting the attachment of 2molecules of FITC on G₄-PAMAM-O-GABA-NH-FITC (FIG. 88).

Synthesis of G₄-PAMAM-O-GABA-Alexa Conjugate

G₄-PAMAM-O-GABA-NH₂ dendrimer was reacted with the Alexa 488 carboxylicacid succinimidyl ester (FIG. 104). The N-succinimidyl activated esterof Alexa 488 couples to the terminal primary amines to yieldamide-linked G₄-PAMAM-O-GABA-Alexa conjugate. The formation of conjugatewas confirmed by HPLC (FIG. 89) using florescent detector (λex=495nm/λem=521 nm). The G₄-PAMAM-O-GABA-NH-Alexa conjugate showed a singlepeak at 15.5 min in the reverse phase HPLC chromatogram. The absence ofany other peak in chromatogram after dialysis of product confirms theabsence of free alexa. Further, the stability of the conjugate in PBS(pH 7.4) after 72 hours was evaluated by HPLC analysis, which showed asingle peak for the conjugate and alexa was not released from theconjugate. The MALDI spectrum showed that the mass of G₄-PAMAM-GABA-NH₂increased from 14,949 Da to 16065 Da confirming the attachment of 2copies of alexa on G₄-PAMAM-O-GABA-NH-Alexa (FIG. 88). The dendrimeralexa conjugate was prepared for enhanced histological visualization ofsamples as confocal imaging causes quenching and also to match with theintensities of other alexa conjugated secondary antibodies.

Transmembrane Transport of G₄-PAMAM Dendrimer

The dendrimer transport and permeability was determined fromchorioamnion i.e. the intact fetal membrane comprising the amnion andchorion together (n=7). The chorion was mechanically stripped off fromthe intact fetal membrane to study the permeability across the amnion(n=7) and the chorion (n=7) separately. The experiments were conductedin dark to avoid quenching of fluorescence. Individual membranes wereused to determine which membrane acts as a rate limiting barrier for thepermeability of molecules based on the size. Usually the permeability ofa molecule directly reflects the interactions of the molecules with thetissue and physiological properties of the tissue. TheG₄-PAMAM-GABA-NH-FITC used for transport and permeability study ishereafter referred to as dendrimer and the unconjugated or free FITC isreferred as FITC.

PAMAM dendrimers are nanosized macromolecules and their size increasesfrom 1.1-12.4 nm as they proliferate from generations 1-10. The Alexaand FITC labeled G₄-PAMAM dendrimers synthesized in the present studyhave a size of 5.6 and 5.4 nm respectively, as seen from the particlesize analysis by DLS. In case of the biological compartments, theepithelium acts as a general barrier for the entry of nanosizedmaterials into the body. The paracellular transport of nanomaterialsacross the epithelium is prevented by the presence of tight junctionsand adherens which have a small gap <2 nm. So far there has been littleor no information on the transmembrane transport of G₄-PAMAM dendrimersacross the human chorioamniotic membrane. In the past,carboxyfluorescein encapsulated liposomes were used to studytransplacental transport and fluorescein has been used to studytransplacental transport in vitro using BeWo (chorionic) cell line.Carboxyfluorescein does not bind to the tissue proteins, it is inert anddoes not undergo biotransformation and its molecular weight is similarto the commonly used therapeutic agents therefore it is considered assuitable marker for transplacental transfer. Literature shows thatfluorescein is established marker for transplacental transfer, hence thefluorophore (FITC and Alexa) tagged G₄-PAMAM dendrimers were used in thepresent study.

As compared to the larger G₄-PAMAM dendrimer molecule, the small FITCmolecule showed a rapid transport across all the three membranes(chorioamnion, amnion and chorion, respectively) in first few hours (in2-3 hours) as seen from FIGS. 90, 91, and 92. The transport of FITC wasfastest across the amnion and a near complete transport(49%)(concentration equilibration) of FITC on the receptor side seemedto occur in 2 hours (FIG. 91B). About 26% of FITC was transported acrossthe chorion in 5 hours (FIG. 92B). The transport of FITC fromchorioamnion was slower than that observed for amnion and chorion, andabout 20% transmembrane transport was seen in first 5 hours (FIG. 90B),while a complete transport occurred after 20 h (FIG. 90A). The transportof G₄-PAMAM dendrimer from all the three membranes across to thereceptor side was negligible (<3%) in the initial few hours (5 hours)(FIGS. 90-92B). The transport of dendrimer did not seem to change withrespect to concentration (3 and 0.6 mg/mL) in first 5 hours and wassignificantly low when compared to FITC. The dendrimer transport forlower concentration (0.6 mg/mL) increased from ≤3% at 5 hours to 8.3% in20 hours for chorioamnion, while for amnion it increased from ≤3% at 5hours to 22% in 20 hours and for chorion in increased from 3% in 5 hoursto 10% in 20 hours. The transport of dendrimer was slowest fromchorioamnion followed by chorion and was relatively faster in amnion. Tomimic the in vivo conditions the transport across chorioamnion isrelevant. The transmembrane transport of the dendrimer seemed toincrease slightly as time progressed (24-30 hours) but substantialamount of dendrimer was not found to transport when compared to FITCalone across all the three membranes as seen from FIGS. 90-92A.Previously, an inverse relation with the molecular weight and thetransport across the BeWo (choriocarcinoma) cell line was observed forvarious markers such as fluorescein, sucrose, dextans and several aminoacids of varying molecular weights. The molecular sieving of the BeWomonolayers seemed to restrict the transport of peptides >1033 Da and theparacellular route was major pathway for transport.

The solute membrane partition coefficient is another parameter thataffects the transport across the biomembrane. The two possible pathwaysfor the solutes to cross the fetal membrane barriers are (a)transcellular route and (b) water filled trans-trophoblastic channels.The hydrophilic molecules are mostly transported thorough the waterfilled channels with the exception of very few hydrophilic solutes whichshow transcellular transport across the human placenta. The Log P valuesfor the G₄-PAMAM dendrimers are negative indicative of its hydrophilicnature, therefore the transcellular mechanism of transport seemsunlikely and the major transport mechanism for these molecules could bethrough the water filled transmembrane channels or pores. Thehistological evaluation was carried out to further evaluate themechanism of transport and biodistribution discussed in subsequentsections. The overall results show that the dendrimer in size range 5-6nm do not cross the chorioamnion appreciably (<3%) in first 5 hours.This, combined with the fact that dendrimers biodistribute relativelyrapidly (with ˜2-3 hours), suggest that dendrimers could be candidatesfor selective topical delivery to the mother without affecting thefetus.

Permeation of G₄-PAMAM (1) and FITC

The transport of molecules across the membranes occurs as a result ofpassive diffusion or active transport. The passive diffusion differsfrom the active process such that the passive diffusion of the compoundthrough the cell membrane is dependant on the concentration gradientwith a constant permeability coefficient. Previously, it has been shownthat the quantity of D-arabinose or carbohydrate transferred across 1cm² of human chorion per unit gradient per unit time can be given by

$\frac{D}{\Delta\; x} = \frac{P}{A}$Where, D is Diffusion coefficient, Δx is the thickness of the tissuestudied, P is the permeability constant and A is the area. When thepermeability coefficient is known it's often used to calculate the otherunknown parameters such as diffusion coefficient (D) or partitioncoefficients (k) or the membrane thickness.

In the current study, the influence of dendrimer size vs the small ofmolecule (FITC) on permeability through the fetal membranes wasevaluated. As per the Fick's law of diffusion, the permeability of thesolute can be given by the equation,

$\frac{P}{\delta} = {\frac{- V}{2{At}}\ln\frac{\Delta\; C_{t}}{C_{0}}}$where C_(t) is the solute concentration in the receptor cell; C₀ is theinitial solute concentration in the donor cell; V is the volume of eachhalf cell; A is the effective permeation area; P is the permeabilitycoefficient; t is the time; and δ is the thickness of the membrane. Theabove equation can be rewritten as

${\ln( {1 - {2\frac{C_{t}}{C_{0}}}} )} = {\frac{{- 2}A}{V}{Pt}}$To determine the permeability coefficient, P, a plot of −V/2A ln(1−2C_(t)/C₀) against t was constructed and linear fitting wasperformed. The slope of the linear portion of the graph yields apermeability coefficient. The thickness of the chorioamnion, chorion andamnion was 0.22 mm, 0.16 mm and 0.05 mm respectively (n=7), as measuredwith the help of Mitutoyo Super Caliper. Table 7 shows the permeabilitycoefficients for G₄-PAMAM-dendrimer and free FITC through the differentmembranes. The FIGS. 93A-C and FIG. 94 show the plots used forcalculation of the permeability and the correlation in all the casesranged from 0.96 to 0.99.

The permeability of FITC (Mw=389 Da) was found to be 1.32×10⁻⁶ (r²=0.97)and 2.26×10⁻⁶ cm²/s (r²=0.99) for the chorion and amnion respectively.Previously, the in vitro permeability of cell free amnion was reportedto be 1.5×10⁻⁶ cm²/s for D-glucose and 2-aminoisobutyrate. Further, thein vitro permeability across chorion for meperidine (Mw=247.33 Da) anddiazepam (Mw=284.7 Da) was reported to be 5.26×10⁻⁶ and 4.51×10⁻⁶ cm²/srespectively. The results for the FITC seem to be within the range tothose reported comparing the molecular weights of these compounds toFITC. The permeability of FITC from chorioamnion was found to be7.93×10⁻⁷ cm²/s. These results show that amnion is more permeable toFITC than the chorion. The permeability of fetal membranes in rhesusmonkey is similar to that of humans and the chorion and chorioamnion inthe rhesus monkey were found to be less permeable than the amnion.Further, previous reports arranged the permeability for water, sodiumions, urea, D-arabiniose and sucrose in the orderamnion>chorion=chorioamnion.

The permeability of small molecule FITC is 100 folds higher from chorionand amnion alone when compared to the permeability ofG₄-PAMAM-dendrimer. When permeability across chorioamnion (intactmembrane) was compared, FITC was found to be 10-fold more permeable thanthe dendrimer (Table 7). The permeability of the compounds was inverselyproportional to their molecular weights. The permeability of dendrimerin the amnion was concentration dependant with a value of 1.86×10⁻⁸cm²/s (r²=0.98) for low concentration and 2.08×10⁻⁷ cm²/s (r²=0.97) forthe higher concentration (Table 7). While in case of the chorioamnionthe lower concentration (0.6 mg/mL) showed a slightly higherpermeability (7.5×10⁻⁸ cm²/s) (r²=0.97) than that exhibited by thehigher (3 mg/mL) concentration (5.8×10⁻⁸ cm²/s) (r²=0.98). On the otherhand, the chorion alone did not show a concentration dependantpermeability where both the high and low concentrations showed apermeability coefficient of 2.94×10⁻⁸ cm²/s (r²=0.96 and 0.97respectively) (FIG. 93C)). In the present study, the amnion and thechorioamnion were able to differentiate between the high and lowconcentrations of the dendrimer unlike the chorion (FIG. 93A-B).Previous reports have shown that the human amnion has better ability todifferentiate between different cations than the chorion, and also theamnion has better differentiating ability towards the transport of smallnon electrolytes and water than the chorion. The order of conductance ofcations by different layers was reported to beamnion=chorioamnion>chorion. These differences are attributed to thelarger intercellular sites in chorion when compared to amnion and hencethe chorion cannot differentiate between the cations. The entrappedwater in the amnion forms an unstirred water layer which itself acts asan effective diffusional barrier to transport of molecules in additionto the amniotic membrane structure and the human amnion cell layer is amore effective diffusion barrier than chorion.

The molecular weight of the G₄-PAMAM-O-GABA-NH-FITC is approximately 40times higher (˜16 kDa) than the molecular weight of FITC (389 Da) andbased on the dendrimer size, its transport is hindered across themembranes. The fetal membranes allow the passage of small molecules(<600 Da) like sodium and glucose by simple diffusion but do not readilypermit the passage of substances of molecular weight >1000 Da. Theamnion and the chorioamnion, behave physicochemically as porous andpartially semipermeable membranes and their cell junctions made ofdesmosomes, gap junctions and occasional tight junctions offeringresistance for paracellular transport. The human chorion is sieve-likemembrane with large water filled extracellular channels and also theintercellular spaces. The transfer by paracellular pathway is moreimportant than the transcellular pathway in the fetal membranes. Theparacellular transfer is dependant on the different pore sizes and thetrophoblast in chorion region has limited number of dilated branchingwide openings with a diameter of 15-25 nm which regulate the overallpermeability. While the non dilated channels in chorion providetransport for the smaller substances having an effective molecularradius under 2 nm, the clefts at the intercellular junctions furtherhave few tight regions of 4.1 nm in diameter restricting the passage oflarge molecules. The size of G₄-PAMAM-Alexa and G₄-PAMAM-FITC was foundto be 5.6 nm and 5.4 nm respectively, and hence their passage couldoccur through the limited dilated openings in chorion.

There is a linear relationship between the rate of transport and theconcentration of dendrimer till 5 hours, which shows that the transfer(<3%) occurs by passive diffusion for all the three membranes in thistime frame. This observation was from both the permeability andtransmembrane transport plots till 5 hours (FIGS. 90-92B and 93A-C). Atlater time points (5 to 30 hours), as seen from the plots oftransmembrane transport (FIGS. 88-90A), the dendrimer with higher (3mg/mL) concentration showed a lower transport as compared to the lowerconcentration (0.6 mg/mL). This suggests that the major pathway fortransport in initial phase (up to 5-6 hours) is passive diffusion but atlater times a saturable process for the transport of higherconcentration is likely, suggesting an additional pathway for transportacross the membrane. Valproic acid uptake (and transport) by thetrophoblast cells is energy dependant (carrier mediated) and wassaturable at higher concentration. Further, despite similar molecularweights, the transport of lipophilic compound was substantially higherthan the hydrophilic compound. In this study, varying the donorconcentration of dendrimer did not lead to a significant change in thepermeability coefficient in the chorioamnion and the chorion (Table 7).However, the permeability through the amnion alone was found to differwith change in concentration of the dendrimer. These findings suggestthat transmembrane transport of dendrimers occurs by paracellular andenergy dependant pathways.

The previous reports on transplacental and transmembrane transport ofmacromolecules like thyrotropin stimulating hormone (TSH), withmolecular weight 28 kDa using a dual chamber was found to be negligibleacross the placenta and fetal membranes. The results of present studyshow that fetal membranes exhibit barrier properties for transmembranetransport based on molecular weight. These results are consistent withthose reported in past. The experimental observation and inferentialevidence suggests that if the drugs are conjugated to the dendrimers orother polymers of large molecular weights, then their transport acrossthe fetal membranes will be restricted due to the larger size inconjugated form and these agents could be used for the selective topicaldelivery in pregnant women without affecting the fetus. It must bepointed out the present measurements of diffusion from a highconcentration water solution across the chorioamniotic membrane in aside-by-side chamber would overestimate the transport, when compared toa topical application, where volume of the body fluids will be presentat relatively lower levels.

Biodistribution of Dendrimer in the Chorioamniotic Membrane

Confocal microscopy was used for histological visualization of thetransport and biodistribution of dendrimer (G4-PAMAM-GABA-NH-Alexa)across the chorioamniotic membrane. FIG. 95A shows the generalmorphology of the human chorioamniotic membrane. The FIG. 95B shows thecontrol membrane (without the treatment with dendrimer) and withnegative controls rabbit isotype and mouse isotype replacing the primaryantibodies showing the nuclei stained blue with DAPI. To identify thedifferent cells and regions in the chorioamniotic membranes they werestained with cytokeratin and vimentin positive. The transport of thedendrimer across the chorioamniotic membrane as a function of time wasinvestigated and the histology data is shown in FIG. 96. The nuclei forall cells are stained blue (by DAPI), the trophoblast cells in thechorion region are stained cytokeratin positive (red) and the stromalcells in the decidua are stained vimentin positive (magenta). Theprogressive advancement of the dendrimer front across the membrane withrespect to the time (30 min to 4 hours) can be visualized from FIG. 96.The dendrimer is mostly confined to the chorionic regions in themembrane as seen from the differential staining for the amnion andchorionic regions (FIG. 96). At early time points, 30 min to 2 hours(FIG. 96 top panel) the dendrimer is mostly seen in the decidual regionand not much has traversed into the trophoblast region (stained red).While at 2.5 to 3.5 hour time points the dendrimer transport hasprogressed slightly further and sparsely the dendrimer can be visualizedin the trophoblast cells in the chorionic region, though most of thedendrimer seems retained in the decidual region (FIG. 96, middle panel).After 4 hours the dendrimer seems to have traversed into the trophoblastregions as seen from the image (FIG. 96, bottom panel extreme left).

It is interesting to note that with the passage of time (30 min to 4hours) the dendrimer progresses gradually across the decidua into thetrophoblast region, however a corresponding increase of the dendrimertransport across the chorion mesoderm, spongy layer, reticular mesh offibroblast layer, amniotic mesoderm or amniotic epithelium is notobserved from the histological evaluation (FIG. 96). In general, thehistology of membranes shows that the dendrimer is not seen in thechorioamniotic mesoderm and amniotic epithelium for the entire timeframe (30 min-4 hours). Its is reported that the human amnion epithelialcells express the multidrug resistant associated proteins (MRPs) whichare responsible for preventing the accumulation of xenobiotics andcontribute for their efflux out of the amnion cells. A similar mechanismwas speculated for the negligible transport of alkaline phosphatase (180kDa) across the amniotic epithelial cells, while the small molecules(<600 Da) were reported to be largely transported by paracellularpathways. The transport experiments showed ≤3% transfer of dendrimerfrom the chorioamnion upto 5 hours. It appears from theimmunofluorescence images that whatever dendrimer traverses acrosschorionic trophoblast region is also transported across the amnionwithout being retained by the amnion cell layer, while the dendrimer ismostly accumulated and retained by the chorionic trophoblast region.

To further evaluate if the dendrimers were taken up by the cells in thechorionic region the histology of membranes was evaluated under highermagnification (63×). The colocalization images (FIG. 97A-B) with eithercytokeratin or vimentin show the internalization of dendrimer in bothcytokeratin positive trophoblast cells and vimentin positive stromalcells. The colocalization of the dendrimer is seen in the nuclei of thetrophoblast cells in chorion (FIG. 97B) and the nuclei and cytoplasm ofstromal cells in decidua (FIG. 97A). Further, the image (FIG. 97A) showsthat the dendrimer surrounds these stromal cells suggesting that bothparacellular and transcellular mechanisms could be responsible fortransport, though passive diffusion seems to be dominant and only asmall fraction of the dendrimer might be internalized in the cells. Asimilar observation was seen for the trophoblast cells where thedendrimer is largely found in interstitial spaces as compared to thatbeing taken up in nuclei (FIG. 97B). Internalization of dendrimers intothe lysozyme and cytoplasm by endocytosis in A549 lung epithelial cellshas been previously reported. Further, colocalization of dendrimers incytoplasm and nucleus of HeLa and cancer cells is known. Also transportof dendrimers by paracellular and transcellular pathway for Caco-2 cellline and microglial cell line is reported in the past. The dendrimer isindeed internalized in some of the trophoblast and stromal cells infetal membranes.

Cellular permeation pathways exist in human fetal membranes and they arecapable of differentiating between different molecular species. Thetransport data showed that the higher concentration of the dendrimer (3mg/mL) at later time points (20-30 hours) did not show theproportionately higher transport across the membranes. This suggeststhat the concentration gradient was not the only driving force for thetransport and there could be a possibility of dendrimer being retainedin the cells. It is possible that the cells are saturated at higherconcentration of dendrimer and hence the correspondingly highertransport at this concentration was not observed. The histology data forlater time point 4 hours showed internalization of dendrimer in moststromal cells. A saturable phenomenon for transport was observed forhigher concentration of valproic acid in trophoblast cells. Thetranstrophoblast transfer of D-glucose and 2-aminoisobutyrate showedboth saturable and non-saturable pathways and accumulation introphoblast cells. These previous results collectively with thetransport data and histological evaluation of immunofluorescent imagessuggest that some dendrimer could be retained intracellularly in thelayers of the chorioamnion membrane. There are reports indicating thatcertain types of particles are accumulated in the placental membranecells rather than crossing the barriers after extended time periods. Thegold nanoparticles 10-30 nm were internalized in the placental cells(trophoblast cells) and traceable amounts were not transported to thefetal side in 6 hours. Also, the energy dependant pathway forinternalization of the small liposomes (70 nm) probably by endocytosisin the placental tissues was reported. Some amount of the liposomes (70nm) was transported by endocytosis to the fetal side. The largemultilamellar liposomes (300 nm) were minimally internalized and theanionic and neutral liposomes were preferentially internalized over thecationic liposomes.

The most significant observation from the present study is that theG₄-PAMAM dendrimers do not cross the intact human fetal membranesignificantly (<3%) in 5 hours, and cross in relatively small amounts(˜10%) over extended time periods up to hours. The dendrimer is mostlyseen retained in chorionic regions. The results show that when comparedto the smaller molecules (e.g. free FITC), which show rapid transportacross the chorioamnion (intact membrane) the G₄-PAMAM dendrimers showedrelatively negligible transport. The strength of this study is that itwas conducted on the fetal membranes of women who underwentcesarean-section delivery and had intact fetal membranes. Thisinvestigation of transmembrane transport of dendrimer from intact fetalmembranes is more relevant to correlate with the transport of dendrimersfrom formulations applied to pregnant women topically on the vaginalmucosa. The polylysine based dendrimers are used as topical microbicidalagents to treat genital herpes and the vaginal gels based formulationsare currently under human clinical trials. Recently, the PAMAMdendrimers were reported to exhibit antimicrobial activity. The presentstudy indicates that these dendrimers could be used as topicalantimicrobial agents or as a component in any intravaginal dosage form(e.g. vaginal tablet, solution or gel) and possibly be used in pregnantwomen without affecting the fetus. These are the preliminary results andfurther extensive investigations are under way.

Conclusions

Selective treatment of the pregnant women without affecting the fetus isalways desired which probes the search for effective drug deliveryapproaches. The transmembrane transport for G₄-PAMAM dendrimer and FITCwas measured across intact human chorioamnion (fetal) membrane andthrough the stripped amnion and chorion membrane individually. Indeed,the G₄-PAMAM dendrimers (Mw ˜16 kDa) tagged with FITC showedsignificantly slower rate of transport across the fetal (chorioamniotic)membranes when compared to the transplacental marker free FITC (Mw ˜389Da). The dendrimer transport was less than ≤3% from all the membranesupto 5 hours and increased slightly in 20 hours, with about 8.3% forchorioamnion (intact membrane), 22% for amnion and 10.5% for chorion,respectively. The transport of FITC was fastest across the amnion withalmost complete FITC seen on the receptor side in 2 hours (49%), about26% in 5 hours from chorion and 20% across chorioamnion in 5 hours,respectively. The biodistribution study showed that the dendrimer ismostly retained in the decidual stromal cells in 30 min to 2 hours. Withprogression in time the dendrimer traverses upto the chorionictrophoblast cells (2.5 to 4 hours). To some extent, the dendrimer isinternalized in nuclei of trophoblast cells and nuclei and cytoplasm ofstromal cells. Largely, the dendrimer is seen in the interstitialregions of stromal and trophoblast cells indicating the passivediffusion as major transport route. The results suggest that dendrimerscould be used as topical antimicrobial agents or as components ofintravaginal dosage forms for selective treatment of pregnant womenwithout affecting the fetus. The overall findings further show thatentry of drugs conjugated to macromolecules would be restricted acrossthe human fetal membrane when administered topically by intravaginalroute.

TABLE 7 Permeation coefficients of G4-PAMAM-O-GABA-NH-FITC (D-FITC)dendrimer and free FITC Permeability coefficients cm²/s CompoundsChorioamnion Chorion Amnion D-FITC 7.5 × 10⁻⁸ 2.94 × 10⁻⁸ 1.86 × 10⁻⁸0.6 mg/mL D-FITC 5.8 × 10⁻⁸ 2.94 × 10⁻⁸ 2.08 × 10⁻⁷ 3 mg/mL FITC 7.93 ×10⁻⁷  1.32 × 10⁻⁶ 2.26 × 10⁻⁶ 0.3 mg/mL

FIG. 104 shows the schematic representation for the synthesis offluorescently labeled G₄-PAMAM-dendrimers; G4-PAMAM-O-GABA-NH-FITC (1)and G4-PAMAM-O-GABA-NH-Alexa (2).

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

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What is claimed is:
 1. A composition comprising a polyamidoamine (PAMAM)dendrimer linked to N-acetyl cysteine, optionally via one or morespacers.
 2. The composition of claim 1, wherein the PAMAM dendrimer is ageneration 3, generation 4, generation 5, generation 6, generation 7,generation 8, generation 9, or generation 10 PAMAM dendrimer.
 3. Thecomposition of claim 2, wherein the PAMAM dendrimer is a generation 4PAMAM dendrimer.
 4. The composition of claim 3, wherein the generation 4PAMAM dendrimer has one or more terminal functional groups selected fromthe group consisting of carboxylic, amine, and hydroxyl groups.
 5. Thecomposition of claim 1, wherein the dendrimer is linked to N-acetylcysteine via one or more spacers.
 6. The composition of claim 5, whereinthe one or more spacers are selected from the group consisting ofN-Succinimidyl 3-(2-pyridyldithio)-propionate, Glutathione,Gamma-aminobutyric acid, and combinations thereof.
 7. The composition ofclaim 1, wherein the dendrimer is linked to N-acetyl cysteine viadisulfide bonds.
 8. The composition of claim 1, wherein the compositionis in an amount effective to treat inflammation or neuroinflammation ina subject in need thereof.
 9. The composition of claim 1, wherein thecomposition is in an amount effective for targeting to activatedmicroglia and astrocytes of the central nervous system.
 10. Thecomposition of claim 1, wherein the composition is in an amounteffective to reduce the level of hydrogen peroxide, and/or nitrite inactivated microglia and astrocytes of the central nervous system. 11.The composition of claim 1, wherein the composition is in an amounteffective to treat and or prevent one or more symptoms of cerebral palsyor white matter injury in the brain.
 12. The composition of claim 1formulated for parenteral, topical or oral administration.
 13. Thecomposition of claim 1 formulated in a form selected from the groupconsisting of hydrogels, nanoparticle or microparticles, suspensions,gels, ointments, powders, tablets, capsules and solutions.
 14. A methodof reducing or preventing inflammation comprises administering aneffective amount of composition according to claim
 1. 15. The method ofclaim 14, wherein the inflammation is chronic inflammation associatedwith heart attack, Alzheimer's disease, congestive heart failure,stroke, arthritis, aortic valve stenosis, kidney failure, lupus, asthma,psoriasis, pancreatitis, allergies, fibrosis, surgical complications,anemia, fibromyalgia, or combinations thereof.
 16. The method of claim14, wherein the inflammation is neuroinflammation.
 17. The method ofclaim 16, wherein the neuroinflammation has pathogenesis associated withactivated microglia and/or astrocytes.
 18. The method of claim 16,wherein the neuroinflammation is associated with cerebral palsy.
 19. Amethod of reducing or preventing microbial growth in a subject in needthereof comprising administering an effective amount of the compositionaccording to claim
 1. 20. The method of claim 19, wherein the method ofreducing or preventing microbial growth comprises administering via aroute selected from the group consisting of vaginal, cervical, andrectal routes.